Detection of molecular interactions by beta-lactamase reporter fragment complementation

ABSTRACT

Methods and compositions for detecting molecular interactions, particularly protein-protein interactions, using at least two inactive, weakly-complementing β-lactamase fragments are provided. The invention allows detection of such interactions in eukaryotic and mammalian cells, in situ or in vitro. Detection of molecular interactions in mammalian cells is not limited to the nuclear compartment, but can be accomplished in the cytoplasm, cell surface, organelles, or between these entities. Methods provided utilize novel compositions comprising fusion proteins between molecules of interest and inactive, weakly-complementing β-lactamase fragments. Association of the molecules of interest brings the corresponding complementary β-lactamase fragments into close enough proximity for complementation to occur and β-lactamase activity to be observed. The invention is useful in the study of protein-protein interactions, functional genomics, agonist and antagonist screening and drug discovery.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority to U.S. Provisional PatentApplication Ser. No. 60/344,757 filed Dec. 26, 2001, the disclosure ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates generally to the field of molecularbiology. More specifically, the invention provides methods andcompositions for β-lactamase-derived reporter systems for detectingmolecular interactions, particularly, but not limited to protein-proteininteractions in mammalian cells.

BACKGROUND OF THE INVENTION

[0003] Homeostasis, proliferation, and differentiation in mammaliancells are regulated by the complex circuitry of interacting proteins.Perturbation of these interactions can lead to disease states such ascancer. Thus, analyzing protein-protein interactions is of extremeimportance to understanding metazoan physiology.

[0004] Protein-protein interactions are involved in every cellularprocess ranging from gene expression and signal transduction to celldivision and differentiation, yet they have been among some of the mostdifficult aspects of cell biology. Standard biochemical methods haveyielded most of the available information about such interactions, butthese assays are often limited by the available reagents such asmonoclonal antibodies for immunoprecipitation, or lack of appropriatecellular context.

[0005] The development of fusion-protein based assays, such as the yeasttwo-hybrid method (Fields, S. & Song, O. (1989) Nature 340, 245-6.),have greatly expanded the potential for studying protein interactions inintact cells. However, this assay relies on the transcription of areporter gene; consequently it is not applicable to studies of thekinetics of protein-protein interactions and is unable to detect theinteraction of compartmentalized proteins such as receptors at the cellsurface. A method based on fluorescence resonance energy transfer (FRET)provided a further advance and is currently one of the most accuratemethods used to monitor dynamic interactions (Adams, S. R., Harootunian,A. T., Buechler, Y. J., Taylor, S. S. & Tsien, R. Y. (1991) Nature 349,694-7.). However, the incremental changes in fluorescence assayed byFRET are small and the stringent steric requirements for detecting theinteracting proteins can restrict the utility of this technique.

[0006] Assays based on the complementation of enzyme fragments fused tointeracting proteins that regenerate enzymatic activity upondimerization are particularly well suited to monitoring inducibleprotein interactions (reviewed in Rossi, F. M., Blakely, B. T. & Blau,H. M. (2000) Trends Cell Biol. 10, 119-122). These systems haveimportant advantages including low level expression of the testproteins, generation of signal as a direct result of the interaction,and enzymatic amplification. As a result, they are highly sensitive andphysiologically relevant assays (Blakely, B. T., Rossi, F. M.,Tillotson, B., Palmer, M., Estelles, A. & Blau, H. M. (2000) NatureBiotechnol. 18, 218-22). Additionally, assays based on enzymecomplementation can be performed in any cell type of interest or indiverse cellular compartments such as the nucleus, secretory vesicles orplasma membrane.

[0007] Systems for the study of protein-protein interactions have beendescribed which utilize two fusion genes whose products reconstitute thefunction of a transcriptional activator. Fields et al., (1989) Nature340:245-247; Bai et al., (1996) Meth. Enzymol. 273:331-347; Luo et al.,(1997) BioTechniques 22(2):350-352. In one fusion gene, a sequenceencoding a first protein is conjugated to a sequence encoding aDNA-binding domain of a transcriptional regulatory protein. In a secondfusion gene, a sequence encoding a second protein is conjugated to asequence encoding a transcriptional activation domain of atranscriptional regulatory protein. The two fusion genes areco-transfected into a cell which also contains a reporter gene whoseexpression is controlled by a DNA regulatory sequence that is bound bythe DNA-binding domain encoded by the first fusion gene. Expression ofthe reporter gene requires that a transcriptional activation domain bebrought adjacent to the DNA regulatory sequence. Binding of the firstprotein to the second protein will bring the transcriptional activationdomain encoded by the second fusion gene into proximity with theDNA-binding domain encoded by the first fusion gene, thereby stimulatingtranscription of the reporter gene. Thus, the level of expression of thereporter gene will reflect the degree of binding between the first andsecond proteins.

[0008] There are several disadvantages associated with the use of theabove-mentioned system. As it is dependent upontranscriptionally-regulated expression of a reporter gene, this systemis limited to the assay of interactions that take place in the nucleus.In addition, the assay is indirect, relying on transcriptionalactivation of a reporter gene whose product is diffusible. Hence, amethod which would allow a direct and immediate examination of molecularinteractions, at the site where they occur, would be desirable.

[0009] A system for detecting protein-protein interactions, not limitedto nuclear interactions, has been described in U.S. Pat. Nos. 5,503,977and 5,585,245. In this system, fusions between potential interactingpolypeptides and mutant subunits of the protein Ubiquitin are formed.Juxtaposition of the two Ubiquitin subunits brought about by interactionbetween potential interacting polypeptides which creates a substrate fora Ubiquitin-specific protease, and a small peptide reporter fragment isreleased. In this system, binding between the potential interactingpolypeptides does not generate any type of enzymatic activity.Therefore, signal amplification is not possible. Additionally, theubiquitin system does not measure in situ activity in intact cells, butrelies on assays of proteolysis in cell-free extracts. What is needed isa sensitive method for examining protein interactions in intact cells inthe relevant cellular compartment.

[0010] The possibility of enzyme fragment complementation withbeta-galactosidase (β-gal) was first shown in prokaryotes. (Ullman, A.et al. J. Mol. Biol. 24, 339-343 (1967); Ullman, A. et al. J. Mol. Biol32, 1-13 (1968); Ullman, A. et al. J. Mol. Biol. 12, 918-923 (1965)).Later studies furthered this technology by extending β-galcomplementation to mammalian cells and showing that it could be used tomonitor inducible protein-protein interactions such as high affinityrapamycin binding proteins and epidermal growth factor (EGF) receptordimerization. (Mohler, W. & Blau, H. (1996) Proc. Natl. Acad. Sci. USA93,12423-12427; Rossi, F., Charlton, C. & Blau, H. (1997) Proc. Natl.Acad. Sci. USA 94, 8405-8410; Blakely, B. et al. (2000) Nat Biotechnol18, 218-222). U.S. Pat. No. 6,342,345 (Blau, et al.) discloses a enzymefragment complementation system using beta-galactosidase (β-gal). Analternative complementation system utilized dihydrofolate reductase(DHFR) fragments to study erythropoietin receptor dimerization. (Remy,I. et al. (1999) Science 283, 990-993; Remy, I. & Michnick, S. (1999)Proc. Natl. Acad. Sci. USA 96, 5394-5399).

[0011] However, both DHFR and β-gal fragment complementation have theirlimitations. DHFR fragment complementation is measured by growth, whereapproximately 25 reconstituted DHFR molecules are required for cellsurvival. Remy, I. et al. (1999) Science 283, 990-993. Thus, the assaydoes not directly monitor real-time protein-protein interactions.Moreover, the DHFR interaction is stoichiometric and does not benefitfrom enzymatic amplification of the signal. Consequently, the signal isweak or requires significant overexpression of the fusion proteins. Inaddition, mammalian cells have endogenous DHFR, which may increase thebackground levels of enzyme activity.

[0012] The β-gal complementation system of U.S. Pat. No. 6,342,345(Blau, et al.) and as described in the literature enzymaticallyamplifies of the signal and can be used to monitor interactions in livecells in real-time. (Rossi, F., Charlton, C. & Blau, H. (1997) Proc.Natl. Acad. Sci. USA 94, 8405-8410; Blakely, B. et al. (2000) NatBiotechnol 18, 218-222). However, β-gal is a large 90 kD molecule whichmay sterically hinder the same interaction it seeks to monitor. Inaddition to the large size of the subunits, β-gal also has thedisadvantage of being a tetrameric complex. The need to form amultimeric complex detracts from the usefulness of this system. β-galalso lacks a cell permeable substrate. Hypotonic shock, used tointroduce the β-gal substrate into cells, is not ideal because it canaffect substrate localization within the cell and can limit the amountof available substrate due to osmotic constraints.

[0013] What is desired is a complementation system that utilizes a smallprotein which has enzymatic activity to allow for signal amplificationand a cell permeable substrate.

SUMMARY OF THE INVENTION

[0014] Class A β-lactamases are particularly attractive candidates foran assay based on enzyme fragment complementation due to the fact thatthey are monomeric and of relatively small size (Philippon, A., Dusart,J., Joris, B. & Frere, J. M. (1998) Cell Mol Life Sci 54, 341-6). Inaddition, β-lactamases have been successfully expressed in prokaryoticand eukaryotic cells, making this system applicable to both classes oforganisms (Moore, J. T., Davis, S. T. & Dev, I. K. (1997) Anal Biochem247, 203-9). An embodiment of the invention relates to the use inmammalian cells of a pair of β-lactamase fragments (α197 and ω198) thatare known to complement well in bacteria when fused to two helices thatform a leucine zipper. Detectable interactions according to thisinvention are not limited to these particular moieties, as interactionsbetween larger proteins are also detectable.

[0015] Extension of the β-lactamase system into mammalian cells providessignificant advantages over other fragment complementation systemscurrently employed (e.g. β-gal (Rossi, F., Charlton, C. A. & Blau, H. M.(1997) Proc Natl Acad Sci USA 94, 8405-10; DHFR (Remy, I. & Michnick, S.W. (1999) Proc Natl Acad Sci USA 96, 5394-9), because the fragments aresmall (<19 kDa), there is no endogenous β-lactamase activity, and ahighly sensitive cell-permeable fluorescent substrate has recently beendeveloped (Zlokainik, G., Negulescu, P. A., Knapp, T. E., Mere, L.,Burres, N., Feng, L., Whitney, M., Roemer, K. & Tsien, R. Y. (1998)Science 279, 84-8). The β-lactamase fragments could be used to monitorinducible interactions in a mammalian cell line measured either byfluorescence microscopy or flow cytometry. The β-lactamase fragmentscould also detect inducible interactions in eukaryotic cells. Further,the observed β-lactamase complementation was a direct measure of enzymeactivity, not dependent on de novo protein synthesis, and generateddetectable signal within minutes of protein dimerization, making itapplicable to the detection of transient protein interactions. Thissystem has broad utility in monitoring protein interactions in diverseintracellular compartments in a range of cell types.

[0016] The present invention provides a novel approach for detectingmolecular interactions in mammalian cells, particularly protein-proteininteractions. In one embodiment, this invention provides a reportersystem based on a first low affinity β-lactamase reporter fragmentcoupled to a first putative binding moiety; and a second low affinityβ-lactamase reporter fragment coupled to a second putative bindingmoiety; wherein the first low affinity β-lactamase reporter fragment iscapable of association with the second low affinity β-lactamase reporterfragment to generate a β-lactamase activity, said association beingmediated by the binding of the first and second putative bindingmoieties. Preferably, the reporter system is used to detectprotein-protein interactions in eukaryotic cells. More preferably, thereporter system is used to detect protein-protein interactions inmammalian cells.

[0017] In another embodiment, the invention provides a method ofdetermining the occurrence of binding between first and second putativebinding moieties in eukaryotic cells, the method comprising: a)providing a reporter system comprising a first component comprising afirst low-affinity β-lactamase reporter fragment, coupled to the firstputative binding moiety; and a second component comprising a secondlow-affinity β-lactamase reporter fragment coupled to the secondputative binding moiety wherein the first low-affinity β-lactamasereporter fragment is capable of association with at least the secondlow-affinity β-lactamase reporter fragment to generate β-lactamaseactivity, said association being mediated by the binding of the firstand second putative binding moieties; b) combining the first componentand the second component; and c) detecting the presence or absence ofthe β-lactamase activity.

[0018] In a further embodiment, the invention provides a method ofscreening for binding of a first binding moiety with members of aplurality of different second putative binding moieties in mammaliancells, the method comprising: a) providing a plurality of reportersystems each comprising: a first component comprising a firstlow-affinity β-lactamase reporter fragment coupled to the first bindingmoiety, and one of a plurality of second components each comprising asecond low-affinity β-lactamase reporter fragment coupled to one of saidplurality of second putative binding moieties, wherein in each of saidsecond components, said second putative binding moiety is differentwherein the first low-affinity β-lactamase reporter fragment is capableof association with the second low-affinity β-lactamase reporterfragment to generate a β-lactamase activity upon the binding of thefirst binding moiety with one of said different second putative bindingmoieties; b) individually combining the first component with each of theplurality of second components in eukaryotic cells to produce aplurality of binding assay samples, each of which includes the firstcomponent and a different one of the second components; and c) detectingthe presence or absence of the β-lactamase activity in each of thebinding assay samples.

[0019] The invention additionally provides nucleic acids encoding fusionproteins including a low-affinity β-lactamase reporter fragment and aputative binding moiety, and the fusion proteins encoded by said nucleicacids. The invention further provides viral vectors comprising nucleicacids encoding such fusions proteins. The invention also provideseukaryotic cells, preferablt mammalian cells, transformed by the nucleicacids and viral vectors described above.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 schematically depicts a protein complementation assayaccording to the invention.

[0021]FIG. 2 shows inducible β-lactamase fragment complementation inC2C12 myoblasts. FIG. 2A is a schematic of the bicistronic, retrovirallyexpressed fusion proteins FKBP12ω198 and α197FRB. FIG. 2B shows animmunofluorescence assay of β-lactamase activity. FIG. 2C shows a FACSanalysis of β-lactamase activity.

[0022]FIG. 3 shows a time course of inducible β-lactamasecomplementation in C2C12 myoblasts. FIG. 3A shows a time course ofrapamycin induced dimerization. FIG. 3B shows a mean fluorescence timecourse. FIG. 3C shows the effect of inhibition of protein synthesis onrapamycin induced complementation.

[0023]FIG. 4 shows the interaction of a membrane-bound and cytoplasmicprotein assayed by β-lactamase complementation in C2C 12 myoblasts. FIG.4A shows the membrane-bound and cytoplasmic fusion proteins which wereco-expressed in C2C12 cells. FIG. 4B shows flow cytometry analysis ofcells expressing fusion constructs.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0024] The present invention provides a broadly applicableprotein-protein interaction biosensor, which has significant advantagesover traditional biochemical as well as existing protein fragmentcomplementation systems. This system should enable the identification ofmolecules that promote or inhibit key protein interactions viahigh-throughput screens in a range of cell types, phyla and species.Further, given its unique properties, β-lactamase may be particularlywell suited to identifying novel protein interactions specific tosubcellular compartments of transformed, proliferating anddifferentiating cell types via an eukaryotic two hybrid assay.

[0025] The present invention provides methods and compositions fordetecting, assaying and quantitating molecular interactions withinmammalian cells and in vitro, through complementation between two ormore low affinity reporter fragments, such as distinct β-lactamasefragments. In a preferred embodiment, protein-protein interactionswithin the mammalian cells are detected and quantitated using themethods and compositions of the present invention. The practice of thepresent invention enables the study of protein-protein interactions andtheir control in living mammalian cells without reliance upon thetranscriptional activation of a reporter gene construct. Association ofthe proteins of interest results directly in enzyme activity and isindependent of other cellular functions. Therefore, the presentinvention provides advantages over other systems currently in use byallowing the detection of complexes that are excluded from the nucleus,and detection of complexes whose formation would inhibit transcription.Furthermore, the present invention allows the detection and localizationof specific binding interactions within eukaryotic cells at differentstages of development and differentiation, and an analysis of theinduction or inhibition of binding interactions in mammalian cells.

[0026] Interactions occurring within the nucleus of the mammalian cell,interactions occurring in the cytoplasm, on the cell surface, within oron the surface of organelles, or between cytoplasmic and surface (eithercellular or organellar) molecules, as well a interactions occurringoutside the mammalian cell, are all capable of being detected in thepractice of the present invention. Thus, the invention surmounts thelimitations associated with previous assays for protein-proteininteractions, which were either limited to interactions occurring in thenucleus, or did not always allow accurate localization of molecularinteractions, and which were not well-suited for detection ofinteractions which resulted in inhibition of transcription ortranslation.

[0027] Protein Fragment Complementation Assays

[0028] Protein fragment complementation assays (PFCA) are used to studyprotein-protein interactions in vitro or in vivo. Protein fragmentcomplementation involves fusing weakly complementing fragments of thesame enzyme to binding proteins of interest. The interaction of thefusion proteins is monitored by enzymatic activity of the reconstitutedfragments in eukaryotic cells. In theory, the non-enzymatic componentsof the fusion proteins force the physical interaction of the enzymaticfragments. Thus the complementation of enzymatic fragments does notdrive, but rather monitors the interaction of other proteins (FIG. 1).When β-lactamase fragments, Δα and Δω, with a breakpoint at glutamicacid 172 are fused to proteins that do not dimerize, their associationis not favored and β-lactamase activity is not detected, as shown inFIG. 1A. When the Δα and Δω β-lactamase fragments are fused to proteinsthat can dimerize, the formation of active β-lactamase is favored, asshown in FIG. 1B.

[0029] The protein fragment complementation assays of the invention havewidespread potential for understanding biological processes as they canbe adapted to high-throughput assays, cDNA screens, and the study ofinducible protein interactions. Such biosensors of protein-proteininteractions should be invaluable in elucidating signal transductionpathways in specific cells (transformed, differentiated, dividing) inresponse to well defined extracellular stimuli such as hormones,cytokines and calcium. Moreover, they can be used to screen formolecules that promote or disrupt such interactions, which could servenot only as invaluable biological tools but also be applied to drugdiscovery.

[0030] Although several systems have been developed that use chimeras ofproteins of interest and enzyme fragments to assess proteininteractions, each has its limitations. For example, in mammalian cellsthe fluorescent signal generated by complementation of dihydrofolatereductase is not amplified enzymatically; thus, only small increments influorescence are achieved (Remy, I. & Michnick, S. W. (1999) Proc NatlAcad Sci USA 96, 5394-9; Remy, I., Wilson, I. A.& Michnick, S. W. (1999)Science 283, 990-3). The β-galactosidase system benefits from enzymaticamplification of its signal; however, the active enzyme is ahomotetramer, and the individual fragments are large (80 kDa), making itlikely that some interactions may be sterically hindered (Blakely, B.T., Rossi, F. M., Tillotson, B., Palmer, M., Estelles, A. & Blau, H. M.(2000) Nat Biotechnol 18, 218-22; Rossi, F., Charlton, C. A. & Blau, H.M. (1997) Proc Natl Acad Sci USA 94, 8405-10).

[0031] The small size, monomeric nature, and availability of acell-permeable fluorescent substrate suggested that a mammalian systembased on the β-lactamase enzyme had the potential to overcome many ofthe limitations of existing systems. Indeed, the β-lactamasecomplementation system described here exhibits an extremely high signalto noise ratio measured in mammalian cells by flow cytometry andfluorescence imaging. In addition, the ability to generate signal withinminutes and the capacity to perform the assay in the absence of de novoprotein synthesis suggests that this system may be ideal for studyinginducible and transient protein-protein interactions in any cell type.

[0032] The β-lactamase System

[0033] β-lactamase fragment complementation seeks to overcome thelimitations of the other protein fragment complementation systems inmammalian cells. TEM-1 β-lactamase is a well-characterized class Aβ-lactamase, which confers resistance to ampicillin by hydrolysis of thebeta-lactam ring. Chaibi, E. et al. (1999) J Antimicrob. Chemother. 43,447-458. The TEM-1 β-lactamase of E. coli is the 264 amino acid productof the ampicillin resistance gene of plasmid pBR322. TEM-1 is thearchetype member of homologous Class A β-lactamases, or penicillinases.

[0034] A β-lactamase system was disclosed in U.S. patent applicationSer. No. 09/526,106. This system was comprised of fragmentscorresponding to breakage of the full-length polypeptide chain at Glu197(Glu172 of the mature protein). Further, the system comprised atripeptide, AsnGlyArg (NGR), inserted between the carboxyl terminus ofthe Δω fragment and the linker. This peptide was selected from a randompeptide library for its ability to preferentially enhance the activityof the enzyme when reconstituted from fragments fused to interactors.

[0035] In this invention, this system was applied to a mammalian cellassay system, specifically the FKBP12/FRAP/rapamycin system. Enhancedcomplementation observed for the β-lactamase system in the presence ofthe NGR peptide led to the applicability of the system in assayingprotein interactions in mammalian cells. Utilizing the inducibleFKBP12-FRB dimerization system, it is not only possible to monitor aninducible interaction in mammalian cells using β-lactamasecomplementation, but also that this assay yields a very robust signal of50-100 fold increase in fluorescence from the responding cellpopulation. This finding, as well as the negligible background observedfrom the expression of the fusion proteins in the absence of adimerizing agent, makes the highly sensitive measurement of proteininteractions using this system readily apparent.

[0036] Properties inherent to the β-lactamase system suggest that itapproaches a physiologically relevant measure of protein interactions inmammalian cells. The α197 fragment is ˜19 kDa whereas the ω198 fragmentis only ˜10 kDa. These values are both smaller than many proteins usedto monitor protein localization such as green fluorescent protein,making it unlikely the fragments will significantly alter the functionof the chimeric proteins being analyzed. The assay can be performed inany cell type and can be used to assay dimerization irrespective ofprotein localization. In addition, the system allows detection ofinteractions in as little as 7.5 min., and that this activity can occurin the absence of de novo protein synthesis demonstrating its utility inthe study of inducible or transient protein interactions.

[0037] Binding Moieties

[0038] Binding moieties which can be assayed for their binding affinitywith each other in mammalian cells include any molecules capable of abinding interaction. The binding interaction between the two or morebinding moieties may be either direct or in the form of a complex withone or more additional binding species, such as charged ions ormolecules, ligands or macromolecules.

[0039] The binding moieties which are attached to the reporter fragmentcan be any of a range of different molecules including carbohydrates,lipids, proteins, and nucleic acids, as well as portions, polymers andanalogues thereof, provided they are capable of being linked to thereporter fragment. Exemplary proteins include members of a signaltransduction cascade, proteins regulating apoptosis, proteins thatregulate progression of the cell-cycle or development of tumors,transcriptional regulatory proteins, translational regulatory proteins,proteins that affect cell interactions, cell adhesion molecules (CAMs),ligand-receptor pairs, proteins that participate in the folding of otherproteins, and proteins involved in targeting to particular intracellularcompartments, such as the Golgi apparatus, endoplasmic reticulum,ribosomes, chloroplasts and mitochondria.

[0040] Other exemplary proteins include protein hormones and cytokines.Cytokines include those involved in signal transduction, such asinterferons, chemokines, and hematopoietic growth factors. Otherexemplary proteins include interleukins, lymphotoxin, transforminggrowth factors-α and β, and macrophage and granulocyte colonystimulating factors. Other proteins include intracellular enzymes suchas protein kinases, phosphatases and synthases.

[0041] Exemplary proteins involved in apoptosis include tumor necrosisfactor (TNF), Fas ligand, interleukin-1β converting enzyme (ICE)proteases, and TNF-related apoptosis-inducing ligand (TRAIL). Proteinsinvolved in the cell cycle include deoxyribonucleic acid (DNA)polymerases, proliferating cell nuclear antigen, telomerase, cyclins,cyclin dependent kinases, tumor suppressors and phosphatases. Proteinsinvolved in transcription and translation include ribonucleic acid (RNA)polymerases, transcription factors, enhancer-binding proteins andribosomal proteins. Proteins involved in cellular interactions such ascell-to-cell signaling include receptor proteins, and peptide hormonesor their enhancing or inhibitory mimics.

[0042] Binding of molecules will depend upon factors such as pH, ionicstrength, concentration of components of the assay, and temperature. Inthe binding assays using reporter systems described herein, the bindingaffinity of the binding moieties in mammalian cells should be highenough to permit forced complementation between the reporter fragments.Non-limiting examples of dissociation constants of the binding moietiesin an assay solution, such as a buffered system or cell interior, are onthe order of less than about 10⁻⁸ M, for example, less than about 10⁻⁹M, or optionally, between about 10⁻⁹ to about 10⁻¹² M, depending uponthe properties of the particular assay system.

[0043] Linking of the Reporter Fragment and the Binding Moiety

[0044] The reporter fragment and one or more binding moieties aregenerally linked either directly or via a linker, and are generallylinked by a covalent linkage. For example, when the reporter fragmentand the binding moiety are proteins, they may be linked by methods knownin the art for linking peptides.

[0045] In one preferred embodiment, the reporter fragment and thebinding moiety comprise a fusion protein including the reporter fragmentwhich is a low binding affinity enzyme complement and the binding moietybeing assayed in mammalian cells. The fusion protein can thus beexpressed from an encoding nucleic acid intracellularly. This system isadvantageous since it permits the detection and quantitation ofprotein-protein interactions in mammalian cells, based on enzymaticcomplementation of the low affinity reporter fragments.

[0046] For example, in the embodiment wherein chimeric fused proteinsare produced intracellularly in mammalian cells, that includes one oftwo complementing low affinity β-lactamase segments and a “test” proteinof interest, the detected β-lactamase activity due to interactionsbetween two chimeric proteins of interest will be proportional to thestrength of the interaction of the non-β-lactamase polypeptidecomponents. Thus, the interaction is driven by the test proteins ofinterest, not the complementing mutants. The enzymatic activity servesas an indicator of that interaction. Another advantage of this system isthat only low levels of expression of the test proteins are required todetect binding.

[0047] The fusion gene constructs preferably are constructed andtransformed into mammalian cells to produce low level expression. Thesystem then permits the monitoring of interactions in a given cell inthe presence of endogenous competing protein partners, where the fusionprotein will function as a “tracer” for the binding/associationreaction. Such a system will not be prone to artifacts arising fromoverexpression of introduced proteins. Reduction in expression of fusiongene constructs can be accomplished by choice of appropriate promoters,ribosome binding sites and other regulatory elements. For example,fusion gene constructs can be introduced into vectors in which they lieupstream of an antibiotic resistance gene whose translation is regulatedby the Encephalomyocarditis virus internal ribosome entry sequence(IRES), and which contain a mutation in the splice donor/acceptorsequences upstream of the ATG sequence responsible for translationalinitiation of the fusion gene. This type of construct results in a lowertranslation efficiency of the first coding sequence in a bicistronicmessage, but does not affect translation of the second (antibioticresistance) sequence, which is solely dependent on the IRES. As a resultof these reduced levels of expression, the frequency of spontaneousinteraction of reporter fragments, which is concentration-dependent,will be significantly reduced.

[0048] Expression of Fusion Proteins

[0049] The invention provides fusion proteins comprising a putativebinding moiety and a low-affinity β-lactamase reporter fragment. Theputative binding moiety may comprise any protein or other molecule whoseability to bind to a second molecule is to be tested. The low affinityreporter fragments comprise β-lactamase fragments capable ofcomplementation with one another to generate β-lactamase activity.

[0050] Fusion proteins comprise a single continuous linear polymer ofamino acids which comprise the full or partial sequence of two or moredistinct proteins. The construction of fusion proteins is well-known inthe art. Two or more amino acids sequences may be joined chemically, forinstance, through the intermediacy of a crosslinking agent. In apreferred embodiment, a fusion protein is generated by expression of afusion gene construct in a cell. A fusion gene construct comprises asingle continuous linear polymer of nucleotides which encodes the fullor partial sequences of two or more distinct proteins in the sameuninterrupted reading frame. Fusion gene constructs generally alsocontain replication origins active in mammalian cells and one or moreselectable markers encoding, for example, drug resistance. They may alsocontain viral packaging signals as well as transcriptional and/ortranslational regulatory sequences and RNA processing signals.

[0051] The fusion gene constructs of the invention are introduced intomammalian cells to assay for binding between the putative bindingmoieties encoded by the fusion gene constructs. The fusion geneconstructs may also contain promoters and other transcriptional and/ortranslational regulatory sequences that are normally associated with thegene encoding the putative binding moiety. The fusion gene constructsmay be introduced into mammalian cells by any method of nucleic acidtransfer known in the art, including, but not limited to, viral vectors,transformation, co-precipitation, electroporation, neutral or cationicliposome-mediated transfer, microinjection or gene gun. Viral vectorsinclude retroviruses, poxviruses, herpesviruses, adenoviruses, andadeno-associated viruses. Particularly preferred in the presentinvention are retroviral vectors, which are capable of stableintegration into the genome of the host cell. For example, retroviralconstructs encoding integration and packaging signals, drug resistancemarkers and one or more fusion genes of interest are useful in thepractice of the invention.

[0052] Different fusion gene constructs encoding unique fusion proteinsmay be present on separate nucleic acid molecules or on the same nucleicacid molecule. Inclusion of different fusion gene constructs on the samenucleic acid molecule is advantageous, in that uptake of only a singlespecies of nucleic acid by a mammalian cell is sufficient to introducesequences encoding both putative binding partners into the cell. Bycontrast, when different fusion constructs are present on differentnucleic acid molecules, both nucleic acid molecules must be taken up bya particular mammalian cell for the assay to be functional. Thus,problems of cell mosaicism are avoided when both fusion gene constructsare included on the same nucleic acid molecule.

[0053] The fusion gene constructs or fusion proteins of the inventionmay be introduced into cultured mammalian cells, mammalian cells invivo, or mammalian cells ex vivo in which it is desired to studyprotein-protein interactions.

[0054] Assays

[0055] The reporter systems disclosed herein may be used to assaybinding interactions of putative binding moieties attached to lowaffinity reporter fragments through complementation between the lowaffinity reporter fragments which produces a detectable signal. Inaddition to testing for direct binding interactions between the putativebinding moieties, interactions dependent upon one or more additionalmolecules or ions may be evaluated. Further, biomolecular interactionsin living animal cells can be evaluated, as well as the influence ofvarious drugs, peptides and pharmaceuticals on these interactions.

[0056] In one embodiment, the binding affinity of one or more putativebinding moieties may be measured by providing a reporter systemincluding one component having one of the moieties bound to a lowaffinity β-lactamase reporter fragment and at least one other componentincluding one other putative binding moiety bound to a second lowaffinity β-lactamase reporter fragment. The binding moieties may bedifferent or the same. In the system, the β-lactamase reporter fragmentsare capable of binding and generating a detectable signal only if theyare brought into proximity by the binding of the one or more putativebinding moieties. The signal can be directly or indirectly detected andquantitated.

[0057] In one embodiment of the invention, protein-protein interactionscan be detected and quantitated. The signal produced by thecomplementing reporter fragments can serve as an indicator of bindingbetween the putative binding moieties, either directly or indirectly viaa third substance. Signals which could be detected include lightemission and absorbance. Exemplary signals include chromogenic,fluorescent and luminescent signals. These signals can be detected andquantitated visually or through the use of spectrophotometers,fluorimeters, microscopes, scintillation counters or otherinstrumentation known in the art.

[0058] Binding of components of the reporter systems disclosed hereinwill depend upon factors such as pH, ionic strength, concentration ofcomponents of the assay, and temperature. Assay solutions can bedesigned and developed for a particular system. The reporter systemsdisclosed herein can be used to conduct assays in systems, such asbuffered cell free extracts of mammalian cells, cell interiors,solutions of cells, solutions of cell lysates, and solutions of cellfractions, such as nuclear fractions, cytoplasmic fractions,mitochondrial fractions, and membrane fractions. Methods for preparingassay solutions, such as enzyme assay solutions, cell extracts, and cellsuspensions, known in the art may be used. For example, physiologicallycompatible buffers such as phosphate buffered saline may be used. Seefor example, the series entitled Methods in Enzymology, Academic Press,New York.

[0059] In one embodiment, the low affinity β-lactamase reporterfragments are capable of complementing one another to form anenzymatically active complex that is capable of catalyzing theconversion of a substrate to a product which is detectable, eitherdirectly or indirectly. In one embodiment, the β-lactamase reportersystem can include two or more components, each of which is a fusionprotein, wherein the fusion proteins each comprise a putative bindingprotein fused to a low affinity β-lactamase reporter fragment. Thus,nucleic acids encoding the fusion proteins can be constructed,introduced into cells and expressed in cells. Alternatively, the boundβ-lactamase reporter units or bound binding moieties can be detecting bydetecting the binding of a labeled specific binding moiety such as anantibody to the bound complex.

[0060] In one embodiment, the low affinity reporter β-lactamase subunitsmay be complementing subunits of β-lactamase which are required toassociate in order to produce a detectable β-lactamase activity. Methodsfor detecting the reaction products of active β-lactamase that have beendeveloped in the art may be used. For example, β-lactamase activity maybe measured by a range of methods including live-cell flow cytometry andhistochemical staining with a chromogenic substrate, such as nitrocefin.Nitrocefin is a cephalosporin which serves as a chromogenic substratefor β-lactamase. Hydrolysis of nitrocefin converts this compound from ayellowish to a pinkish color. U.S. Pat. No. 5,955,604 (Tsien et al.)discloses fluorescent substrates of β-lactamase and is incorporatedherein in its entirety.

[0061] Vital substrates for β-lactamase, which can be used in mammaliancells, are also encompassed by the invention. For example, CCF2/AM canbe used for live cell sorting of cells expressing β-lactamase.

[0062] Detection of Binding Events

[0063] The methods disclosed herein enable the detection andquantitation of binding events in cell lysates, as well as in intactmammalian cells. Thus, interactions between fully folded proteins aredetectable, and co-translational expression of the binding moieties isnot necessary for binding to be detected. In the practice of theinvention, the reaction product may be detected indirectly, for example,through immunological techniques, such as immunofluorescent labeling.

[0064] Fluorescence imaging has been used to study the intracellularbiochemistry of mammalian cells. A fluorescent indicator for theadenosine 3′,5′-cyclic monophosphate (cAMP) signaling pathway has beendescribed in which the sensor is a cAMP kinase in which the catalyticand regulatory subunits each are labeled with a different fluorescentdye, such as fluorescein or rhodamine, capable of fluorescence resonanceenergy transfer in the holoenzyme complex. A change in shape of thefluorescence emission spectrum occurs upon cAMP binding, and thereforeactivation of the kinase can be visualized in cells microinjected withthe labeled holoenzyme. Adams et al., Nature, 349: 694-697 (1991). Thissystem is limited by the fact that it requires microinjection, and apreferred distance between the labeled units for energy transfer tooccur.

[0065] Substrates for β-lactamase have been described in the art whichinclude a fluorescent donor moiety and a quencher, which include anattached group which makes them permeable through mammalian cellmembranes, wherein the attached group is hydrolyzed off after thesubstrate enters the cell. Fluorescence energy transfer between thedonor and quencher is monitored as an indicator of β-lactamase activity.This system also can be used in a reporter gene assay using cellscontaining β-lactamase reporter genes functionally linked to a promoter.Substrates for β-lactamase are described in WO 96/30540 published Oct.3, 1996, and U.S. Pat. No. 5,955,604 the disclosures of which isincorporated herein.

[0066] Protein-protein interactions can be measured in a mammalian cellreporter system which includes one or more fusion proteins. The fusionproteins each include a putative binding protein coupled to a lowaffinity β-lactamase reporter fragment. For intracellular expression ofthe fusion proteins, one or more fusion gene constructs are preparedwhich include sequences encoding the fusion protein(s). The fusion geneconstructs may be introduced into mammalian cells by methods availablein the art, including, but not limited to, viral vectors,transformation, co-precipitation, eclectroporation, neutral or cationicliposome-mediated transfer, microinjection or gene gun.

[0067] A variety of cell-based assays can be conducted using the cellscontaining the fusion gene constructs. Binding of the putative bindingmoieties on the fusion proteins expressed in the cells can be confirmedby detecting the signal produced by the β-lactamase reporter fragmentsundergoing forced complementation.

[0068] The fusion gene constructs may also contain promoters and othertranscriptional and/or translational regulatory sequences that arenormally associated with the gene encoding the putative binding moiety.This permits the study of physiologically-relevant levels of theputative binding proteins in vivo, in contrast to systems in which testproteins are overexpressed. Further, this permits the study ofnaturally-occurring changes in levels of binding activity over time andcan reveal the effects of endogenous or exogenous substances on bindinginteractions.

[0069] The methods and compositions of the invention can also be used tostudy other molecules which influence the interaction of two putativebinding partners in mammalian cells. Proteins, peptides, nucleic acids,carbohydrates, lipids, ions, small molecules, synthetic compounds orother substances (either endogenous to the cell or exogenously added)may act as either agonists or antagonists of a binding interaction. Bymeasuring the effect of such molecules on, for example, β-lactamaseactivity produced by mammalian cells containing two or more fusionsrepresenting a particular pair of test proteins, agonist or antagonistactivity of such molecules can be determined. Use of the methods andcompositions of the invention will allow high-throughput assays to becarried out to test for agonists or antagonists of a particular bindinginteraction. Such high-throughput assays will be especially valuable inscreening for drugs that influence medically-relevant protein-proteininteractions.

[0070] Putative binding partners, or putative binding moieties,according to the invention, can include molecules which do not normallyinteract with each other, but which each interact with a third moleculesuch that, in the presence of the third molecule, the putative bindingpartners are brought together. Thus, substances which influence aninteraction between putative binding partners include those whichstimulate a weak interaction between putative binding partners, as wellas one or more molecules which mediate interaction between moleculeswhich do not normally interact with each other. In addition, substanceswhich influence an interaction between putative binding partners caninclude those which directly or indirectly affect an upstream eventwhich results in association between the putative binding partners. Forexample, if phosphorylation of one of the putative binding partnersendows it with the capacity to associate with another of the putativebinding partners; substances which influence the interaction of theputative binding partners include those which directly or indirectlyaffect a kinase activity.

[0071] Assays can be developed for mammalian cells as disclosed hereinto examine the effect on intermolecular interactions of a variety ofcompositions including drugs such as antipyretic and anti-inflammatorydrugs, analgesics, antiarthritics, antispasmodics, antidepressants,antipsychotics, tranquilizers, antianxiety drugs, narcotic antagonists,anti-Parkinsonism agents, cholinergic antagonists, chemotherapeuticagents, immunosuppressive agents, antiviral agents, parasiticides,appetite suppressants, antiemetics, antihistamines, antimigraine agents,coronary vasodilators, cerebral vasodilators, peripheral vasodilators,hormonal agents, contraceptives, antithrombotic agents, diuretics,antihypertensive agents, cardiovascular drugs, opioids, and vitamins.

[0072] Protein-protein interactions mediated by a third molecule can bedetected and quantitated in mammalian cells. The kinetics of bindingalso can be studied. Such systems are described in Examples 1-4 below,wherein β-lactamase fusion proteins are used to monitor therapamycin-mediated interaction of the FKBP12 and FRAP proteins. Belshaw,P. J. et al., Proc. Natl. Acad. Sci. USA, 93: 4604-4607 (1996); Brown etal., Nature 369: 756-758 (1994); Chen, et al., Proc. Natl. Acad. Sci.,USA, 92:4947-4951 (1995); and Choi, J. et al, Science, 273:239-242(1996). For example, kinetics of binding can be determined by measuringβ-lactamase activity at different times following addition of rapamycinto cultures of cells expressing fusions of FKBP12 and FRAP to twocomplementing, low affinity β-lactamase fragments (e.g., Δα and Δω). Adose-response curve can also be obtained, in which the extent ofbinding, as measured by β-lactamase activity, is determined as afunction of rapamycin concentration.

[0073] The reporter system can also be designed with controls to permitthe quantitation of the expression level of the β-lactamase fusionproteins in mammalian cells. This will make it possible to control forpotential differential expression of the two (or more) fusion proteins.For example, a peptide tag for which well-characterized monoclonalantibodies are available may be fused in frame at the C-terminus of eachβ-lactamase fragment. Different tags, such as flag and myc may be usedfor Δα and Δω, to allow differential detection of the two mutants evenwhen coexpressed in the same cells. In parallel with the determinationof β-lactamase activity in the lysates of these mammalian cells, anELISA assay can determine the precise amount of each β-lactamase fusionprotein in the same lysates. First, a polyclonal anti-β-lactamaseantiserum may be used to immobilize the antigens. Then the monoclonalantibody directed against the appropriate tag followed by anenzyme-linked anti-mouse secondary antibody may be used to quantify theamount of the β-lactamase fusion protein of interest. Such an approach,employing well-characterized techniques, should allow a determination ofthe expression levels of each fusion protein. This modification will beuseful where the attached tag does not impair the binding of the proteinor the ability of the reporter fragments to complement.

[0074] Applications of the Invention

[0075] As will be apparent to one of skill in the art, the inventionallows a broad range of studies of protein-protein and other types ofmulti-molecular interaction to be carried out quantitatively orqualitatively in mammalian cells. In what follows, non-limiting examplesof different applications of the invention are provided.

[0076] The observation that levels of β-lactamase activity in thepresence and absence of forced complementation can be distinguished byFACS (FIG. 2), suggests that the methods of the invention can be used toscreen for new binding partner(s) for a given target protein inmammalian cells. In this embodiment, the target protein, fused to aweakly-complementing β-lactamase fragment is stably expressed in awell-characterized mammalian cell line. Expression libraries containingcDNAs fused to a weakly-complementing β-lactamase mutant are introducedinto these cells using, for example, retroviral vectors (e.g., Kitamuraet al., Proc Natl. Acad. Sci. USA 92:9146-9150 (1995)) or any othermeans of gene transfer known in the art. Vectors expressing geneproducts that interact with the target protein are isolated byidentifying β-lactamase-positive clones. An advantage of this system isthat the screen can be carried out in any type of mammalian cell,regardless of the cell's milieu of endogenous (and potentiallycompeting) proteins. A further possibility for this type of system isthat the target protein can be localized to a specific cellularcompartment, with the aim of identifying proteins involved ininteractions restricted to that particular location.

[0077] The assays and methods of the invention can also be carried outin the presence of extracellular signaling molecules, growth factors ordifferentiation factors, peptides, drugs or synthetic analogs, or thelike, whose presence or effects might alter the potential forinteraction between two or more given proteins in a particular mammaliancell type.

[0078] Detection of molecular interactions, using the methods andcompositions of the invention, is not limited to those occurring in thenucleus, nor is it limited to intracellular interactions in mammaliancells. For instance, interactions involving surface receptors can bedetected in the practice of the invention. In one embodiment, theinvention provides new techniques for detecting ligand-induceddimerization of surface receptors in mammalian cells. Dimerization, orhigher order oligomerization, of cell surface receptors is often aprerequisite for receptor activation and ensuing signal transduction.For example, the binding of epidermal growth factor (EGF) to itsreceptor stabilizes the dimerization of the receptor and leads toactivation of its tyrosine kinase activity. Schlessinger et al. (1992)Neuron 9:383-391; Ullrich et al. (1990) Cell 61:203-212; and Weiss et al(1997) Curr. Opin. Genet. Dev. 7:80-86. Example 11, infra, discloses theuse of β-gal complementation to monitor membrane receptor dimerizationin mammalian cells.

[0079] By combining the methods and compositions of the invention withstate-of-the-art methods for construction of high-titer, high-complexitycDNA libraries in retroviruses (e.g., Pear et al., (1993) Proc. Natl.Acad. Sci. USA 90:8392-8396), it will be possible to identifyinteraction partners of a specific test protein in mammalian cells(i.e., perform functional genomics at the protein level). For thisapplication, construction of cDNA libraries in retroviral vectorswherein the cDNA coding sequence is fused to a sequence encoding a lowaffinity β-lactamase reporter fragment will be used. A sequence encodinga binding protein of interest will be fused to a low affinityβ-lactamase reporter fragment in a first retroviral vector. In a secondseries of retroviral vectors, a second complementing low affinityreporter β-lactamase subunit will be fused to a variety of differentproteins that will be tested for their ability to bind to the protein ofinterest. Testing will be conducted by co-infection of mammalian cellswith the first and one of the series of second retroviral vectors. Thosetest proteins which are capable of binding to the protein of interestwill allow detection of a reporter signal in cells in which they areco-expressed with the protein of interest. This application will also beuseful in screening for agonists and antagonists of medically-relevantprotein interactions.

[0080] The use of fluorescence-activated cell sorting techniques isparticularly well-suited to this embodiment of the invention. Forexample, β-lactamase-positive mammalian cells which contain cDNAsexpressing gene products that interact with the target protein willgenerate a signal that will allow such cells to be purified bycell-sorting techniques. Such cDNAs could be delivered, for example,using retroviral vectors that allow introduction of high complexity cDNAlibraries with high infection efficiency.

[0081] In one embodiment of the invention, mammalian cells in which aprotein encoded by one of the series of second vectors is able tointeract with the binding protein of interest encoded by the firstvector are detected and isolated by flow cytometry orfluorescence-activated cell sorting (FACS). Methods for flow cytometryand FACS are well-known in the art; e.g., Nolan et al. (1988) Proc.Natl. Acad. Sci. USA 85:2603-2607; Webster et al., Exp. Cell Research,174:252-265 (1988); and Parks et al. (1986) in The Handbook ofExperimental Immunology, (eds. Weir, D. M., Herzenberg, L. A.,Blackwell, C. C. & Herzenberg, L. A.), Blackwell, Edinburgh, 4thedition, pp. 29.1-29.21. In this way, clones of cells in which bindingoccurs can be isolated and propagated for further study. This aspect isparticularly suited for studies of developmental mechanisms, wherein itis possible to select a population of mammalian cells in which aparticular developmentally-relevant interaction has occurred and studythe further development of that cell population, while at the same time,studying the further development of the cells in the population in whichthe interaction has not occurred, for comparison. In a similar fashion,the practice of the invention makes it possible to isolate and/or studythe further development of mammalian cells exhibiting interactionsinvolving protein such as transcriptional regulatory proteins,translational regulatory proteins, DNA replication proteins, mRNAsplicing proteins, proteins involved in signal transduction, proteinsinvolved in cell-cell and cell-substrate adhesion (for example, cellmovement, axon guidance and angiogenesis), oncogene products, tumorsuppressors, proteins involved in cell-cycle control and viral proteins,such as those involved in regulation of viral replication, virus-hostinteractions and virus assembly, and proteins which are subunits,crosslinkers, modifying agents or molecular motors within thecytoskeleton of cells.

[0082] For a given target protein whose gene is capable of being fusedto a low-affinity complementing reporter β-lactamase subunit, it ispossible to identify known and heretofore unknown proteins or otherendogenous or extraneous substances with which it interacts, by usingthe compositions and methods of the invention. In like manner, for asequence which encodes a protein of unknown function, such as may beobtained from a nucleic acid sequence database, (or a plurality ofsequences such as a cDNA library) the practice of the invention allowsone to identify molecules with which the encoded protein interacts. Theidentity of the interacting molecule(s) is likely to provide informationwith respect to the structure and/or function of the unknown protein.Thus, the practice of the invention will likely aid in theidentification and characterization of newly-discovered proteins andprotein-coding nucleic acid sequences.

[0083] In another aspect of the invention, a shotgun approach to theidentification of protein-protein interactions can be taken bygenerating a first set of constructs which will express the encodedproducts of one cDNA library fused to a first low-affinity complementingβ-lactamase subunit and a second set of constructs which will expressthe encoded products of a second (or the same) cDNA library, fused to asecond low-affinity complementing β-lactamase subunit. Co-expression ofthe two sets of constructs and selection of cells in whichcomplementation occurs will allow the isolation of clones and theidentification of cDNAs which encode interacting partners. One or bothof the interacting partners may be known; alternatively, both of theinteracting partners may represent heretofore unidentified proteins. Ifboth partners are known, new information about their binding specificitymay be obtained. If one partner is known, it may provide information onthe function of the unknown binding partner. If neither are known, theobservation that they interact may assist in the eventual identificationof one or both of the interacting pair.

[0084] The invention may be applied to studies of the mechanisms thatregulate either homo- or hetero-dimerization of specific molecules,including high efficiency screening to identify synthetic or naturallyoccurring compounds capable of influencing such dimerization.

[0085] The invention can be used for investigations relating to thelocalization of specific complexes within intact mammalian cells, orintact animals. Types of mammalian cells which can be used are primaryor established cell lines and other types of embryonic, neonatal oradult cells, or transformed cells (for example, spontaneously- orvirally-transformed). These include, but are not limited to fibroblasts,macrophages, myoblasts, osteoclasts, osteoclasts, hematopoietic cells,neurons, glial cells, primary B- and T-cells, B- and T-cell lines,chondrocytes, keratinocytes, adipocytes and hepatocytes.

[0086] It is also possible, through practice of the invention, to devisesystems for regulation of enzyme activity by regulating the associationof complementing β-lactamase fragments in a mammalian cell. This aspectof the invention has potential applications to human therapy, as amethod to regulate the enzyme-driven conversion of pro-drugs into theiractive forms.

[0087] Processes involving molecular interactions, particularlyprotein-protein interactions, which can be studied in the practice ofthe invention include, but are not limited to, transcription,translation, replication, mitosis, growth control, progression andregulation of the cell-cycle, apoptosis, cell-cell, cell-substratum andcell-ligand interactions, intracellular signal transduction cascades,oncogenesis, cell lineages, and embryonic development. Examples of cellligands include leptin and growth factors such as epidermal growthfactor (EGF), nerve growth factor (NGF), platelet-derived growth factor(PDGF), and insulin-like growth factors I and II (IGF-I and IGF-II),transforming growth factors α and β (TGF-α and TGF-β), endorphins andendorphin receptors, prostaglandins and their receptors, cytokines andtheir receptors, neurotransmitters and their receptors, adrenergicreceptors, and cholinergic receptors. Receptors which could interactwith ligands include EGF, NGF, and PDGF receptors and leptin receptors.

[0088] Additional interactions that can be studied by the practice ofthe invention include interactions involved in cell metabolism and cellstructure. These include, but are not limited to, interactions that areinvolved in energy metabolism or which establish or modify the structureof the membranes, cytoplasm, cytoskeleton, organelles, nuclei, nuclearmatrix or chromosomes of cells. Interactions among constituents of theextracellular matrix, or between constituents of the extracellularmatrix and cells, can also be studied with the methods and compositionsof the invention.

[0089] The invention will be further understood by the followingnon-limiting examples.

EXAMPLES Example 1

[0090] Inducible β-Lactamase Complementation in Mammalian Cells.

[0091] A reporter system using β-lactamase complementation to evaluateprotein-protein interactions was constructed. Experiments were designedto test whether the β-lactamase fragments in conjunction with the NGRpeptide could be used to monitor an inducible protein interaction inmammalian cells (FIG. 2). The well characterized inducible interactionof FKBP12 and FRB was used as a model system (Chen, J., Zheng, X. F.,Brown, E. J. & Schreiber, S. L. (1995) Proc Natl Acad Sci USA 92,4947-51; Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith,C. T., Lane, W. S. & Schreiber, S. L. (1994) Nature 369, 756-8; Ho, S.N., Biggar, S. R., Spencer, D. M., Schreiber, S. L. & Crabtree, G. R.(1996) Nature 382, 822-6; Belshaw, P. J., Ho, S. N., Crabtree, G. R. &Schreiber, S. L. (1996) Proc Natl Acad Sci USA 93, 4604-7; Choi, J.,Chen, J., Schreiber, S. L. & Clardy, J. (1996) Science 273, 239-42; thedisclosures of which are incorporated herein). FKBP12 (FK506 BindingProtein 12) binds FRB (the FKBP12 binding domain of FRAP) only in thepresence of the pharmacological agent rapamycin, an interaction thatincreases with the dose of rapamycin. Rapamycin is a small cellpermeable molecule that can be added directly to the culture mediumresulting in heterodimerization of FKBP12 and FRB. Since rapamycin isunable to bind two FKBP12 molecules at the same time and FRAP only bindsrapamycin within the FKBP12-rapamycin complex, only heterodimers formupon rapamycin treatment. (Ho, S. N. et al., Nature, 382:822-826 (1996),the disclosure of which is incorporated herein).

[0092] Two fusion proteins were constructed using flexible linkers(Gly₄Ser)₃: (i) FKBP12 was fused to the N-terminus of the ω198 fragmentand (ii) FRB was fused to the carboxy-terminus of the α197 fragmentcontaining the NGR peptide (FIG. 2A). FIG. 2A shows a schematic of thebicistronic, retrovirally expressed fusion proteins FKBP12ω198 andα197FRB with selectable markers for hygromysin (hygro^(R)) and neomycin(neo^(R)) driven by an internal ribosome entry sites (IRES). Ψdesignates the viral packaging signal and LTR marks the long terminalrepeats. The bacterial signal sequence from each of the β-lactamasefusion fragments was removed. The fusion constructs were expressed usingpWZL retroviral vectors that encode proteins conferring resistance tohygromycin or neomycin. The pWZL vectors were selected for use becausethey are expressed at relatively low levels; in these vectors the splicedonor/acceptor is deleted, resulting in reduced translation efficiencyin mammalian cells compared to other retroviral vectors such as MFG(Rossi, F., Charlton, C. A. & Blau, H. M. (1997) Proc Natl Acad Sci USA94, 8405-10; Riviere, I., Brose, K. & Mulligan, R. C. (1995) Proc NatlAcad Sci USA 92, 6733-7). Thus, these vectors avoid vast overexpressionof proteins and more closely approximate physiological levels.

[0093] A stable cell line, containing the FKBP12ω198-hygro andα197FRB-neo constructs, was established through retroviral infection ofmouse myoblast C2C12 cells, and subsequent antibiotic selection. Cellsfrom this population were treated with 50 nM rapamycin for 2 hours andassayed for β-lactamase activity using the fluorogenic CCF2/AM substrate(Zlokarnik, G., Negulescu, P. A., Knapp, T. E., Mere, L., Burres, N.,Feng, L., Whitney, M., Roemer, K. & Tsien, R. Y. (1998) Science 279,84-8). The intact CCF2/AM substrate when excited by a UV wavelength of409 nm emits at 520 nm (green), whereas upon cleavage by β-lactamase itemits at 447 nm (blue). FIG. 2B shows an immunofluorescence assay ofβ-lactamase activity. C2C12 cells expressing the FKBP12ω198 and α197FRBfusions were loaded with the cell permeable CCF2/AM substrate, in theabsence (upper panel) and presence (lower panel) of rapamycin (2 hours),and then imaged by fluorescence microscopy. Green is indicative ofintact substrate, whereas blue indicates cleaved substrate. As shown inFIG. 2B (upper panel), the cells expressing the fusion proteins appeargreen in the absence of rapamycin, indicating that little or no cleavageof the substrate has occurred. However, upon exposure to rapamycin thesubstrate is cleaved, shifting the fluorescence from green to blueindicating reconstitution of β-lactamase activity (FIG. 2B, lowerpanel). These results revealed that inducible dimerization of FKBP12 andFRB could lead to the complementation of the β-lactamase fragmentsresulting in functional β-lactamase activity in mammalian cells.

[0094] These data were confirmed by performing a quantitativemeasurement of β-lactamase activity by flow cytometry (FACS) using theCCF2/AM substrate (FIG. 2C). FIG. 2C shows a FACS analysis ofβ-lactamase activity. Cells with and without rapamycin treatment (2hours) were trypsinized, loaded with CCF2/AM substrate and assayed byflow cytometry. Increases in cascade blue fluorescence are indicative ofβ-lactamase activity (log scale). Upper panel—β-lactamase staining ofcells expressing wild-type β-lactamase; middle panel—untransduced cellsstained with the CCF2 substrate; lower panel—cells expressing theβ-lactamase fusion constructs with and without rapamycin. Dimerizationof the fusion constructs induced by rapamycin causes a 50-100 foldincrease in cascade blue fluorescence from the responding population.The histograms of the cells that stably expressed FKBP12ω198 and α197FRBin the absence of rapamycin (lower panel) overlapped with and were notsignificantly different from untransduced negative control cells (middlepanel). By contrast, following exposure of the cells harboring theβ-lactamase fragments to rapamycin for 2 hours, enzyme activity wassubstantially induced and an increase in fluorescence of 50-100 foldabove background was evident. Two features of these data areparticularly noteworthy: (1) the almost undetectable background activityresulting from complementation in the absence of rapamycin and (2) themarked increase (orders of magnitude) in the signal generated by thecomplementation.

[0095] In this experiment, 20% of the cells expressing wild-typeβ-lactamase did not stain positive for β-lactamase activity even thoughthe cells were kept in continuous drug selection to ensure retention ofthe virus containing the wild-type β-lactamase gene. A similarpercentage of non-responding cells (˜23%) can be seen in the populationof cells expressing the chimeric β-lactamase proteins in the presence ofrapamycin either by flow cytometry or fluorescence imaging. Thisphenomenon was also noted in the original study describing the CCF2/AMsubstrate with similar ratios, 80% responding and 20% not responding,suggesting that this may be a feature of the substrate stainingprocedure itself (Zlokamik, G., Negulescu, P. A., Knapp, T. E., Mere,L., Burres, N., Feng, L., Whitney, M., Roemer, K. & Tsien, R. Y. (1998)Science 279, 84-8).

Example 2

[0096] Time Course of Inducible β-Lactamase Complementation in C2C12Myoblasts

[0097]FIG. 3A shows a time course of rapamycin treatment in C2C12 cellsexpressing the chimeric β-lactamase fusion proteins that demonstratedthe ability to distinguish quantitatively the responding from thenon-responding population. The FKBP12ω198-α197FRB cell line was assayedfor β-lactamase activity by FACS. Cells were stained with the CCF2/AMsubstrate, treated with rapamycin, and assayed over time. In FIG. 3A,the gate is represented in the center of each plot and the percentage ofcells falling within this region are shown in red. When a gate was drawnaround the cells expressing the fusion constructs prior to induction(time zero), it only included 0.9% of the cells. Notably, 10% of thecells were positive at 7.5 min after rapamycin addition, and at one hour76% of the population stained positive for β-lactamase activity, i.e.,most if not all, of the cells capable of responding. Longer rapamycintreatment did not significantly increase the numbers of positive cells.

[0098] The time course was rapid and began to plateau within 15 min.This is most clearly evident when the data from a FACS analysisperformed in triplicate are presented as mean fluorescence (FIG. 3B).FACS data (from A) represented as mean cascade blue fluorescence werecalculated in triplicate and graphed over time. Following rapamycinaddition, a response was seen as early as 7.5 min after rapamycintreatment and was 70% maximal within 15 min demonstrating that using abulk assay for fluorescence, the generated signal is also detectablewithin minutes of induced complementation. These kinetics aresignificantly faster than those reported for other systems utilizing theFKBP12-FRB proteins to induce dimerization (Otto, K. G., Jin, L.,Spencer, D. M. & Blau, C. A. (200 1) Blood 97, 3662-4; Muthuswamy, S.K., Gilman, M. & Brugge, J. S. (1999) Mol Cell Biol 19, 6845-57), whichdemonstrates the high specific activity of the complemented enzyme andthe extreme sensitivity of the system.

[0099] The rapid kinetics of β-lactamase reconstitution upon addition ofrapamycin suggested that de novo protein synthesis might not benecessary. To test this possibility, β-lactamase activity was assayed inthe presence of the protein synthesis inhibitors puromycin andcycloheximide at concentrations of 100 μg/ml for 2 hours prior to theaddition of rapamycin. Neither of these inhibitors significantly alteredthe amount of complementation observed relative to the controls,indicating that de novo protein synthesis is not necessary forβ-lactamase complementation (FIG. 3C). As shown in FIG. 3C,α197FRB-FKBP12ω198 cells were treated with either puromycin orcycloheximide (100 ug/ml) for two hours before addition of rapamycin (1hour). The cells were stained with the CCF2/AM substrate and assayed byflow cytometry. The mean fluorescence for the cascade blue channel wascalculated in triplicate and graphed on the Y-axis. Many inducibleprotein-protein interactions have been documented to occur on atimescale of seconds to minutes. The data shown here suggest that theβ-lactamase system has the potential to monitor not only rapid, butpossibly also transient protein-protein interactions.

Example 3

[0100] Detection of Constrained Protein-Protein Interactions inMammalian Cells

[0101] β-lactamase fragment complementation in an inducibleprotein-protein interaction in mammalian cells was carried out usingfusion proteins expressed in myoblast cells. These fusion proteins wereforced to interact by an inducer, and analyzed in the presence of afluorescent substrate for β-lactamase activity. The negative control forβ-lactamase activity was the same cell line expressing fusion proteinsin the absence of inducer. The “signal” of β-lactamase activity of cellsexpressing fusion proteins that had been induced to interact wascompared to the “noise” of the negative control. The positive controlfor β-lactamase activity was a cell line expressing wild-typeβ-lactamase. β-lactamase activity in cells expressing the fusionproteins was analyzed following addition of the inducer at differenttime points to determine the time course and maximum signal.

[0102] Cell surface mediated signal transduction events often requirethe interaction of a membrane associated protein, such as atransmembrane receptor with a cytoplasmic protein. In order to testwhether the β-lactamase system is capable of monitoring interactions insuch a constrained configuration, a model membrane bound protein wasconstructed that would interact with a cytoplasmic protein only in thepresence of an inducer. For this purpose, a tripartite fusion constructwas generated comprised of the extracellular and transmembrane regionsof EGFR (tEGFR, see Blakely, B. T., Rossi, F. M., Tillotson, B., Palmer,M., Estelles, A. & Blau, H. M. (2000) Nat. Biotechnol 18, 218-22)) thatanchored the protein to the plasma membrane fused to the FKBP12ω198chimera (FIG. 4). FIG. 4A shows a diagram depicting the membrane-boundand cytoplasmic fusion proteins which were co-expressed in C2C12 cells.The tEGFR (truncated Epidermal Growth Factor Receptor) was used totether the FKBP12ω198 to the plasma membrane. This fusion protein wasco-expressed with a cytoplasmic α197-FRB chimera, as previouslydescribed.

[0103] Cells expressing these constructs were assayed by FACS forinduction of β-lactamase activity in the presence of rapamycin (FIG.4B). FIG. 4B shows cells expressing the fusion constructs, in thepresence (upper panel) or absence (lower panel) of rapamycin (1 hour),were assayed by flow cytometry. The magnitude of β-lactamasecomplementation observed when proteins are in a constrained,membrane-anchored conformation is comparable to that observed when theproteins are freely expressed in the cytoplasm (compare FIG. 3A and FIG.4B).

Example 4

[0104] Retroviral Vectors for Expression of β-Lactamase in MammalianCells

[0105] The β-lactamase system was combined with theFKBP12/FRAP/rapamycin system to generate mammalian cell expressionsystems as follows:

[0106] MMLV-based retroviral vectors with antibiotic resistance toeither G418 or hygromycin were selected. Retroviral vectors were chosenbecause 1) they stably integrate into the DNA of mammalian cells and 2)they use a bicistronic mRNA to couple antibiotic resistance with uptakeand expression of the fusion construct. The construct was a MoloneyMurine Leukemia Virus (MMLV)-based retroviral vector that has been shownto efficiently transduce C2C12 myoblast cells (Kinsella, T. & Nolan, G.(1996) Hum. Gen. Ther. 7, 1405-1413).

[0107] To create the β-lactamase fusion proteins for retroviralexpression in mammalian cells an oligonucleotide encoding a GS-linker,

[0108] 5′TCGAGGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCG G, was andinserted into the Xho I/Sal I site of both pWZL-neo and pWZL-hygro. Theα197-NGR fragment was amplified by PCR from plasmid FHT 4002A1 usingprimers

[0109] 5′CTCGAGCACCCAGAAACGCTGG and 3′GTCGACTTCCCGCCCATTTTC G. The ω198fragment was amplified by PCR from using primers

[0110] 5′CTCGAGGGAGTGCAGGTGGAAACC and 3′CTCGACTTCCAGTTTTAGAAGC. The α197fragment was cloned into the XhoI site of pWZL-GS-Neo and the ω198fragment was cloned into the SalI site of pWZL-GS-Hygro. FRBcorresponding to amino acid residues 2025-2114 of human FRAP was clonedas an XhoI/SalI fragment into the SalI site of pWZL-α197-GS-Neo. Thefull length coding sequence of FKBP12 was cloned as a SalI/XhoI fragmentinto the XhoI site of pWZL-GS-ω198-hygro. The tEGFR corresponding to AA1-655 (Blakely, et al. (2000) Nat Biotechnol 18, 218-22) was cloned asan NcoI/BamHI fragment into the pWZL-FKBP12ω198-hygro vector. Thewild-type β-lactamase was expressed from a pWZL vector also encodingpuromycin resistance.

Example 5

[0111] Retroviral Production, Infection, and Mammalian Cell Culture

[0112] The ecotropic ΦNX packaging cell line (P. L. Achacoso and G. P.Nolan, unpublished) was transiently transfected with the proviralconstructs using FuGENE transfection reagent (Boehringer Mannheim,Indianapolis, Ind.). The virus-containing supernatant from thetransfected cells was removed 48-72 hours later and applied to C2C12myoblasts cells which are a well-characterized, fast-growing cell line(Blau, H., Chiu C. & Webster, C. (1983) Cell 32, 1171-1180). Polybrenewas added to a final concentration of 8 μg/ml (Sigma, St. Louis, Mo.).Singly and doubly infected cells were selected by antibioticresistances. Transduced cells were selected and maintained in theappropriate antibiotic (G418, hygromycin, puromycin or neomycin;Invitrogen, Carlsbad, Calif.) at a concentration of 1 mg/mL. C2C12myoblasts were grown in DMEM (Invitrogen) 20% FBS. Cells were treatedwith 50 nM rapamycin unless otherwise stated. The selected cells wereexpanded as populations for testing.

Example 6

[0113] β-Lactamase Assayed by CCF2/AM Staining, Immunofluorescence andFACS Analysis in Mammalian Cells

[0114] The cell lines described above were assayed using a fluorescenceactivated cell sorter (FACS). With FACS, individual, mammalian cells canbe characterized for beta-lactamase activity. Data were collected on amodified Facstar plus (Becton Dickinson, Franklin Lakes, N.J.) withMoFlo electronics (Cytomation, Fort Collins, Colo.). Cells weretrypsinized, washed twice in PBS, incubated with the fluorescentsubstrate CCF2/AM (Aurora Biosciences, San Diego, Calif.) for 1 hour,then washed twice in a PBS 5% FBS solution. In some embodiments, 10,000events were collected for each sample. Cells were excited with a Kryptonlaser (406 nm) and emission data for the samples were collected at420-460 nm (Cascade Blue filter) and 500-590 nm (Cascade Yellow filter)using a Becton Dickinson FACS machine. A decrease in the intensity ofgreen fluorescence and an increase in the intensity of blue fluorescenceindicated β-lactamase activity. Quantification of β-lactamase activityas the ratio (mean blue fluorescence in sample cell line)/(mean bluefluorescence in negative control cell line)+/− standard deviationindicated the signal-to-noise ratio of β-lactamase fragmentcomplementation in the system.

[0115] In an uncleaved CCF2/AM substrate, fluorescence resonance energytransfer between the donor fluorophore (coumarin), excited by an outsidesource, and the acceptor fluorophore (fluorescein), results in energyemission in the green portion of the spectrum. Cleavage of substrate byβ-lactamase prevents excitation of the donor fluorophore by FRET andresults in energy emission in the blue spectrum. The acceptorfluorophore, however, remains in the cell, and thus, would besusceptible to excitation by an outside source. Emission data from cellsexcited by the 407 nm Krypton laser are collected in Cascade Blue (CasB)between 420 nm and 460 nm and in Cascade Yellow (CasY) between 500 nmand 590 mn. Excitation of acceptor fluorescein (FITC) by the 488 nmArgon laser generates an emission spectra with a peak at 525 that wouldbe collected through the CasY filter between 500 nm and 590 nm.

[0116] To assay β-lactamase in C2C12 myoblasts, the CCF2/AM substrate(Aurora biosciences, San Diego, Calif.) was used at a finalconcentration of 2 uM in DMEM with 2.5 mM probenecid. Cells were washed,once in PBS then incubated for 30 min with the CCF2/AM substrate at aconcentration of 3×10⁵ cells/ml. The plate was washed 3 times in PBS andvisualized with a β-lactamase filter set (Chroma TechnologiesBattleboro, VT: excitation 405+/−10 nm, 425 dichroic mirror, 435 nm LPemission).

Example 7

[0117] Tripartite Fusions for the Quantitation of Protein-ProteinInteractions.

[0118] To permit protein interactions to be studied in a quantitativemanner in the system described in the above Examples and to control foreffects on either the binding ability of the binding moiety or thecomplementing ability of the reporter fragments resulting from bothactivities being present in a single fusion protein, additionalmodifications can be made to monitor the expression of the components ofthe system. In the above described system, the β-lactamase fusionproteins will be expressed from the same viral promoter, however, forsome proteins, it is possible that their expression level will beinfluenced by the specific fusion partner. In particular, some proteinsor domains could affect the stability or conformation of the β-lactamasedomain. As a result, differences in the ability of the test proteins(the putative binding moieties) to complement one another could beobserved that are not based on a physiological mechanism.

[0119] In order to avoid these problems, fusions containing threecomponents (β-lactamase fragment, FKBP12 or FRAP, and a test protein)can be constructed. The most N-terminal component can be a test protein,followed by FKBP12-Δω or FRAP-Δα. The presence of the FKBP12 and FRAPportions would allow rapamycin-mediated dimerization of these fusions,and the efficiency of β-lactamase complementation in the presence ofrapamycin would likely be dependent on the FKBP12/FRAP/rapamycininteraction. The absolute values of β-lactamase activity obtained bysimple coexpression (in the absence of rapamycin) of fusions containinga fixed protein of interest and different interacting partners can bedetermined. In parallel samples, β-lactamase activity can be measuredupon induction of complementation with a fixed amount of rapamycin. Theratio between the β-lactamase activity obtained in the absence or in thepresence of rapamycin would indicate the relative ability of thedifferent protein pairs to interact with each other. An added advantageof this approach would be that the presence of the FKBP12 and FRAPdomains provide a flexible hinge between the β-lactamase fragments andthe putative binding moieties that are being analyzed. This reduces thepossibility of interference between β-lactamase and the proteins ofinterest. Furthermore, it allows direct testing of the functionalintegrity of the β-lactamase components in the fusions without the needfor recloning into more efficient viral vectors.

[0120] tetR-FKBP12-Δω or tetR-FRAP-Δα tripartite fusions can also beused. Coexpression of these constructs, in which dimerization is drivenby the tetracycline repressor (tetR) protein (Hinrichs, W. et al.,Science, 264:418-420 (1994), the disclosure of which is incorporatedherein), would yield β-lactamase positive cells. This result wouldindicate that functional tripartite fusions can be constructed, in whichthe dimerization of the most N-terminal peptide component canefficiently drive complementation of the C-terminal β-lactamase fragmentpolypeptides.

Example 8

[0121] Dimerization of Myogenic Regulators Using Complementingβ-Lactamase Fusion Proteins

[0122] The β-lactamase complementation system can be used to assay forthe dimerization and nuclear translocation of HLH proteins(helix-loop-helix proteins, Murre et al. (1989) Cell 56:777-783)including activators of muscle-specific proteins (myoD, myogenin, myf5,MRF-4), inhibitors of myogenesis (Id, Mtwist, I-mf) and ubiquitousE2A-type proteins (E47, E12, HEB).

[0123] In a first step, a myoD-Δα-β-lactamase (myoD-Δα) fusion constructand a E12-Δω-β-lactamase (E12-Δω) fusion construct are engineered inselectable retroviral vectors, as described above for FRAP-Δα andFKBP12-Δω. The two constructs can be transduced into C2C12 myoblasts.Following selection with the appropriate drugs for cells which expressboth constructs, β-lactamase activity can be quantitated using thechromogenic or fluorimetric assay described above. β-lactamase activitywould indicate that heterodimerization of the fusion proteins isoccurring in this cell type. If β-lactamase activity is detected,individual cells can be analyzed using a fluorescent CCF2/AM stain inorder to determine if the heterodimers are present in the nucleus.Wild-type β-gal can be specifically directed to and detected in thenucleus by inclusion of a nuclear localization sequence (nls) (see e.g.,Hughes and Blau, Nature, 345:350-352 (1990)).

[0124] Inclusion of an “nls” sequence in a β-lactamase hybrid proteincan allow direction and detection of the β-lactamase hybrid protein inthe nucleus. Knowledge of the site of localization in the cytoplasm ornucleus would aid in assessing the function of the protein interactions,e.g. sequestration and inhibiting activity, or promoting activity. Thismethod would permit visualization of fluorescent markers of myogenesis,such as desmin, and creatine kinase, in correlation with thelocalization of β-lactamase, using the sensitive CCF2/AM substratedescribed above.

[0125] All fusion constructs between myogenic regulators andcomplementing β-lactamase fragments described in the following sectionsmay be tested in a muscle cell where heterodimerization of theendogenous myogenic regulator is known to occur. In addition, thefollowing controls also may be performed. The myoD-Δα construct may becotransduced into the cell with FKBP12-Δω, and the E12-Δω construct maybe cotransduced with FRAP-Δα. This combination of constructs shouldresult in no β-lactamase activity, unless some unusual mechanism existsin the particular cell type being tested that enhances complementationof the weakly complementing β-lactamase peptides independent ofheterodimerization of the non-β-lactamase parts of the molecule. TheFRAP-Δα and FKBP12-Δω may also be cotransduced and cells treated withrapamycin as a positive control for complementation in each cell type.Cells in high serum medium (growth medium) and cells in low serum medium(differentiation medium) should/will give different results.

Example 9

[0126] In vivo Assay for the Effect of Growth Factors and Substrates onHeterodimerization and Homodimerization.

[0127] Using the constructs described above in Example 8, C2C12myoblasts can be transduced with one of the myogenic HLH fusionconstructs and the E12-Δω construct. Although C2C12 cells will alreadycontain endogenous myogenic HLH proteins and E12, the chimericconstructs will act as a “tracer” to measure the extent ofheterodimerization. Transduced cells then may be stimulated to eitherdifferentiate or proliferate by changes in serum levels or the additionof growth factors (TGF-β, bFGF, IGF-I and IGF-II) in the presence orabsence of substrates such as fibronectin or laminin. β-lactamaseactivity can be measured as a function of time. Rapid changes inβ-lactamase activity after growth factor stimulation may suggest a moredirect mechanism of action of a given extracellular signal on theformation of specific heterodimers. Slower changes may indicate that theextracellular signal acts indirectly, for example by up-regulating theexpression of a competing factor which can sequester one or both fusionproteins. Changes in β-lactamase activity may be correlated with theexpression levels of known inhibitors of differentiation such as Idproteins, measured by Northern blot in parallel samples. A comparison ofthe kinetics of changes in β-lactamase activity obtained with each pairof test proteins in parallel experiments will indicate whether specificMRFs (muscle regulatory factors, Yun et al. (1996) Curr. Opin. CellBiol. 8:877-879; and Cossu et al. (1996) Trends Genet., 12:218-223) orinhibitors differ in their ability to respond to extracellular signals.When a growth factor or substrate capable of influencing heterodimerformation (or nuclear translocation) is identified, the experiments arerepeated in other, non-myogenic cell types. The analysis of the effectof a specific growth factor in different cell types would indicatewhether the intracellular components of the corresponding signaltransduction pathway are tissue-specific.

[0128] These studies in tissue culture cells would permit the relativeaffinity and compartmentalization of specific protein partners underconditions of growth and differentiation, and subsequently in responseto known signal transducers, to be evaluated. The interactions of thesefactors may be tested in a relevant physiological background incompetition with the prevalent endogenous components present in the cellat the time. Most analyses of the interactions of myogenic factorsperformed thus far have been carried out in vitro, in purified systems,or in yeast (Benezra et al., Cell, 61:1213-1230 (1990); Lassar et al.,Cell, 66:305-315 (1991); Hu et al., Mol. Cell. Biol., 12:1031-1042(1992); Chen et al., Cell, 86:731-741 (1996); and Spicer et al.,Science, 272:1476-1480 (1996). The relatively low sensitivity of thebiochemical methods used to directly detect interactions in mammaliancells, such as immunoprecipitation or activation of a reporter geneconstruct, required high levels of protein and overexpression of theconstruct, usually obtained by transient transfection, levels that couldpotentially force an interaction due to increased concentration. Themethods disclosed herein permit protein-protein interactions that arefunctionally relevant at different points in the myogenicdifferentiation pathway to be studied. Clearly, the extracellular andintracellular milieu determines the stoichiometry and abundance of thethese proteins at different times. As a result, competition of differentproteins for the same dimerization partners, cofactors, and kinases orphosphatases in signal transduction pathways could have significanteffects on which complexes actually form in intact cells. To assess thenature of such endogenous interactions, low expression levels are neededin order not to alter the levels inherent to the cell and characteristicof the “competitive” environment at a given time.

[0129] Advantageously, high-level expression of the introduced proteinsis not required in the systems described herein in order to assess theprotein-protein interactions of interest. Indeed, by contrast withtransient transfection assays or even most retroviral vectors withstrong promoters and high translation efficiencies, the systemsdisclosed herein provide levels that should not perturb the naturalendogenous physiological levels of the proposed test proteins in thecell.

Example 10

[0130] Analysis of Inhibitory and Myogenic HLH Proteins in Mice.

[0131] The heterodimerization of inhibitory and myogenic HLH proteins inmice may be mapped. Mtwist and I-mf have been shown to inhibitmyogenesis in mammalian tissue culture systems. In addition, they havebeen proposed to act via direct physical association with myogenic HLHproteins (Hebrok et al., Dev. Biol., 165:537-544 (1994); Rohwedel etal., Exp. Cell Res., 220:92-100 (1995); Chen et al., Cell, 86:731-741(1996); Spicer et al., Science, 272:1476-1480 (1996)). Duringembryogenesis, Mtwist is expressed throughout the epithelial somite andis later excluded from the myotome (Fuchtbauer, Dev. Dyn., 204:316-322(1995); and Stoetzel et al., Mech. Dev. 51:251-263 (1995)). AlthoughI-mf expression has not been analyzed at early stages of somatogenesis,at 11.5 days post-coitum I-mf is highly expressed in the sclerotome butis excluded from the myotome (Chen et al., Cell, 86:731-741 (1996)).Thus, based on their expression domains in the embryo, these factors arethought to be critical for spatial and temporal restriction of themyogenic program in early development.

[0132] Further support for this hypothesis derives from analyses ofmyf5/lacZ embryos in which the myf5 coding region has been targeted andreplaced by lacZ. Using β-gal as a marker of the myf5 expressionpattern, cells expressing myf5 are detected in the presomatic mesoderm,where Mtwist is also expressed (Fuchtbauer, Dev. Dyn., 204:316-322(1995); and Stoetzel et al., Mech. Dev. 51:251-263 (1995)), long beforethe onset of myogenesis (Cossu et al., Trends Genet., 12:218-223(1996)). Later in development, myf5 and myoD are co-expressed togetherwith Mtwist in the somite before the formation of a distinct myotome.Ott, et al., Development, 111: 1097-1107 (1991); Fuchtbauer, Dev. Dyn.,204:316-322 (1995); Stoetzel et al., Mech. Dev. 51:251-263 (1995); andCossu et al., Trends Genet., 12:218-223 (1996)). These cells do notexpress other detectable myogenic markers (Ott, et al., 1991). Thus, thereporter systems disclosed herein may be used to determine if the myf5and MyoD proteins in these cells are maintained in an inactive state byinteraction with Mtwist and/or I-mf in heterodimers. At subsequentstages of development, Mtwist and I-mf are expressed in most of thenon-myogenic mesoderm, where the expression of myogenic factors isexcluded. Smith et al., J. Cell Biol., 127:95-105 (1994); Fuchtbauer,Dev. Dyn., 204:316-322 (1995); Stoetzel et al., Mech. Dev. 51:251-263(1995); and Chen et al., Cell, 86:731-741 (1996). Possibly Mtwist andI-mf are involved in the creation of a sharp border between the myotomeand the adjacent tissues at this stage.

[0133] The reporter systems disclosed herein permit detailed studies ofthe interactions between myogenic inhibitors and activators in vivoduring embryonic development which can provide novel insights into thecomplex process of patterning during somatogenesis. Such studies are notlimited to mice and can easily be performed in C. elegans, Drosophila,Xenopus, zebrafish and other experimental organisms. To date, amethodology that allows visualization of protein complexes in situ inthe embryo has not been available. As a result, no definitive evidenceis available as to when and where during embryonic developmentinteractions of such HLH heterodimers might occur.

Example 11

[0134] Detection of HLH Heterodimers in Mouse Embryos

[0135] The β-lactamase complementation assay is well-suited for thedetection of protein-protein interactions in vivo. Myf5-Δα, MyoD-Δα andMtwist-Δω fusion proteins may be constructed. Mediation of β-lactamasecomplementation with these fusion proteins may be tested in the courseof performing the experiments described above. Using well-establishedtransgenic technology (Thomas and Capecchi, Nature, 324:34-38. (1986);and Capecchi, Science, 244: 1288-1292 (1989)), mouse lines may begenerated in which one of the myf5, MyoD or Mtwist alleles has beenreplaced with the corresponding fusion protein. Thus myf5-Δα, MyoD-Δαand Mtwist-Δω fusion proteins will be expressed under the control oftheir endogenous promoters. The expression of the test protein can beverified in these mice. The Mtwist-Δω transgenic mouse may then becrossed with the myf5-Δα, and the MyoD-Δα transgenic mouse lines, and ineach case the offspring may be analyzed in order to identify thosecarrying both of the fusion proteins. β-lactamase activity should onlydevelop in those cells of the embryo in which Mtwist-Δω physicallyassociates with the myf5-Δα or the MyoD-Δα fusion proteins. Thisanalysis allows mapping when and where during embryonic developmentMtwist is actually interacting with myf5 and MyoD to repress themyogenic phenotype.

Example 12

[0136] Targeting Strategy and Engineering of Necessary Constructs

[0137] The myf5-Δα fusion protein coding sequence may be inserted intothe myf5 locus so that it will be expressed under the control of theendogenous myf5 regulatory elements. An insertion of wild type β-gal inthe myf5 locus resulting in a fusion with the ATG of myf5 has been shownto reproduce faithfully the expression pattern of the endogenous gene. Asimilar strategy may be employed using β-lactamase. The targetingconstruct is based on the published myf5/lacZ targeting construct(Tajbakhsh and Buckingham, Proc. Natl. Acad. Sci. USA, 21:747-751(1994); Tajbakhsh et al., Neuron, 13:813-821 (1994); and Tajbakhsh etal., Nature 384:266-270 (1996)), but with the following differences: (1)The fusion protein contains the complete myf5 coding sequence fused tothe Δα β-lactamase. (2) The fusion protein coding sequence is followedby a neomycin resistance gene flanked by FRT sites (FLP recombinasetargets). This allows G418 selection of ES cells that have taken up andintegrated the targeting construct. (3) A diphtheria toxin expressioncassette is located 5′ of the region of homology with the myf5 mousegenomic DNA. During homologous recombination, strand exchange will occurwithin the homology region and as a result the diphtheria toxinexpression cassette will be excluded following integration (Capecchi,Science, 244: 1288-1292 (1989)). Clones resulting from randomintegration rather than homologous recombination retain diphtheria toxinexpression and will be selected against during culture, because theywill die. The surviving clones are characterized by PCR, and theappropriate integration of the construct in the myf5 genomic locus isconfirmed by Southern blot.

[0138] Subsequently, the neomycin selection cassette is removed using amodified version of a previously described technique (Fiering et al.,Genes Dev., 9:2203-2213 (1995)). Briefly, a plasmid expressing abicistronic message containing FLP recombinase, an Internal RibosomalEntry Site (IRES) and GFP is transiently transfected into the ES cellclones. GFP positive cells are clonally sorted using the fluorescenceactivated cell sorter (FACS). In these cells, FLP deletes the sequencesbetween the two FRT sites, and only the β-lactamase coding sequenceremains in the ES cell genome. Aliquots of the sorted clones are testedfor sensitivity to G418, and in the sensitive clones the accuratedeletion of the neomycin cassette is confirmed by PCR and Southernblotting. This approach, which eliminates the selectable marker, avoidsinterference between the exogenous promoter driving the selectablemarker and the endogenous regulatory sequences as described (Olson etal., Cell, 85:1-4 (1996)).

[0139] Targeting constructs for MyoD and Mtwist have also been described(Rudnicki et al., Cell, 71:383-390 (1992); Chen and Behringer, GenesDevel., 9:686-699 (1995)) and the relevant constructs may be producedfor each. Based on these available reagents, and following the schemeproposed above for the myf5-Δα strategy, vectors to target (Chen andBehringer, Genes Devel., 9:686-699 (1995)) MyoD-Δα and Mtwist-Δω fusionsinto the endogenous MyoD and Mtwist loci of ES cells may be constructed.In each case, an ES cell line syngeneic to the available genomic DNAhomology regions in the targeting construct are used, as straindifferences are known to reduce the frequency of homologousrecombination. The same FLP-mediated excision methodology used for themyf5 “knock in” described above is applied to the deletion of theneomycin resistance markers from the targeted MyoD and Mtwist loci. This“in-out” strategy ensures that the fusion protein coding regions areunder the control of the endogenous regulatory elements and associatedwith minimal extraneous flanking DNA sequences.

Example 13

[0140] Monitoring EGF Receptor Dimerization in Mammalian Cells

[0141] The regulation of the epidermal growth factor (EGF) receptorsignaling pathway and other receptor signaling pathways which actthrough receptor dimerization can be studied using the methods of theinvention for monitoring protein-protein interactions at the membrane oflive mammalian cells. Chimeric proteins containing the extracellular andtransmembrane domains of the EGF receptor, fused to weakly complementingβ-lactamase fragments can be expressed in myoblasts. Treatment of thecells with EGF will result in chimeric receptor dimerization as assessedby a rapid increase in β-lactamase enzymatic activity. The use of such asystem might be used to study aspects of receptor signaling such asfeedback mechanisms in which tyrosine kinase activity of the dimericreceptor inhibits further dimerization of the receptor.

[0142] Construction of chimeric receptors The weakly complementing Δαand Δω deletion mutants of β-lactamase can each be linked to apolypeptide sequence containing the extracellular and transmembranedomains of the human EGF receptor to form chimeric receptor molecules.The chimeric receptors lack the cytoplasmic domain, and attendanttyrosine kinase activity, of the native receptor. Constructs containingthe appropriate β-lactamase-EGF receptor fusions can be made usingstandard molecular biological cloning methods. see Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, New York, N.Y. (1989)).

[0143] DNAs encoding the chimeric receptors can be inserted intoretroviral vectors also encoding a selectable marker. For the constructcontaining the EGF receptor-Δα fusion, the selectable marker can be theneo gene, encoding G418 resistance; while the EGF receptor-Δω fusion canuse hygromycin resistance. Plasmids can be transfected into φNX cellsusing Lipofectamine (Life Technologies), and virus-containingsupernatant can be harvested 48-72 hours later. C2F3 mouse myoblasts(Rastinejad et al. (1993) Cell 72:903-917) maintained in DME with 20%fetal bovine serum (FBS) in 10% CO₂, can be infected by overnightincubation in the viral supernatant. Cells containing both constructscan be selected in 1 mg/ml G418 plus 1 mg/ml hygromycin, and maintainedin 400 μg/ml of each antibiotic.

[0144] EGF treatment and FACS analysis. Cells can be treated with mousesalivary gland EGF (Sigma) at 100 ng/ml and in some experiments they canbe treated with tyrphostin AG1478 (Calbiochem) at 100 nM. Following alltreatments, cells can be rinsed with phosphate buffered saline (PBS),trypsinized, and resuspended in PBS+5% FBS. CCF2/AM can be loaded intothe cells. Cells will be kept on ice until analysis on the cell sorter,which can be conducted 1 to 2 hours after trypsinization.

[0145] The chimeric receptor can be detected by immunofluorescence usinga monoclonal mouse anti-human EGF receptor antibody diluted 1:100 (cloneEGFR1, Dako) and either phycoerythrin-labeled horse anti-mouse IgG(Vector) or fluorescein-labeled goat anti-mouse IgG (Cappel) diluted1:100. Cells can be trypsinized and stained in PBS+5% FBS. For eachsample, FACS analysis data can be collected for 5000 cells. Cells can becloned on a Becton-Dickinson FACS Vantage and analyzed on aBecton-Dickinson FACScan at the Stanford Shared FACS Facility. Dataanalysis can be facilitated by FlowJo software (Tree Star, Inc.). Meanfluorescence data can be adjusted for autofluorescence by subtractingthe mean fluorescence of untransduced cells loaded with CCF2/AMsubstrate.

[0146] Receptor dimerization assay. The two chimeric DNAs can each becloned into retroviral vectors encoding selectable markers andtransduced into the C2F3 mouse myoblast cell line. After selection withG418 and hygromycin, β-lactamase enzyme activity can be monitored usingthe fluorescence activated cell sorter (FACS) to measure the product ofa fluorogenic substrate. In the absence of EGF, the population oftransduced cells would be expected to consist of a mixture of cells withlow and high levels of β-lactamase activity, which would not beunexpected given that the EGF receptor is capable of dimerizing in theabsence of EGF. Gadella et al. (1995) J. Cell Biol. 129:1543-1558.Following stimulation of the population of cells with EGF many of thecells would be exhibited to show increased β-lactamase activity. UsingFACS analysis with an antibody specific to the human EGF receptor,clones can be isolated and screened for low background levels ofβ-lactamase activity in the absence of EGF, and increased levels ofβ-lactamase activity in the presence of EGF. The effect on EGF receptordimerization of other EGF-like growth factors that bind and activate theEGF receptor, such as TGF-α, heparin-binding EGF-like growth factor, andbetacellulin; and EGF-like factors, such as heregulin α, that actthrough related receptors other than the EGF receptor can also bestudied. Beerli et al. (1996) J. Biol. Chem. 271:6071-6076. Dimerizationcan be expressed as the mean fluorescence or β-lactamase activity of thecells.

[0147] Time-course of EGF Receptor dimerization. In order to follow thefate of receptor dimers over time, cells from the an isolated clone canbe cultured in media containing EGF for 0 to 24 hours and then analyzedby FACS. The time course of dimerization can then be studied.Concurrently, a measurement of the levels of the chimeric receptor onthe cell surface by immunofluorescence using FACS can be used todetermine whether the amount of chimeric receptor on the cell surfaceremains essentially constant over the period that dimerization occurs.

[0148] Feedback regulation of EGF Receptor dimerization. This inventioncan be used to monitor feedback regulation of the EGF receptor. Thecomplementation of β-lactamase activity can be used as a measure ofdimerization. The effect of continued EGF treatment of the cells couldbe measured. By measuring the effect of continued application of EGF onEGF-mediated dimerization of the chimeric receptor, one can determine iffeedback regulation of EGF receptor signaling is occurring. Forinstance, resistance to dimerization of the EGF chimeric receptordespite continued application of EGF might indicate that signalingthrough the endogenous wild-type EGF receptors in the cells inhibitsdimerization of the chimeric receptor. This possibility could then befurther tested by using an inhibitor of the EGF receptor such as AG1478,a highly specific inhibitor of the EGF receptor tyrosine kinase.Levitzki et al. (1995) Science 267:1782-1788.

[0149] Accordingly, cells expressing chimeric receptor can be treatedwith EGF overnight, and then retreated with EGF or tyrphostin and theextent of dimerization can be determined by measuring β-lactamaseactivity.

[0150] Thus, the methods and compositions of the invention can be usedto monitor EGF receptor dimerization in live cells. Such experimentswould be expected to yield information on regulation of receptordimerization by phosphorylation. Such a system would also afford ascreening method for the identification of agents that might affectreceptor dimerization.

[0151] The kinetics of complementation reflect the kinetics ofassociation of the binding partners. The time course of dimerization ofthe EGF receptor chimera can be compared to the time course of theinteraction of FRAP and FKBP12. β-lactamase complementation can be usedto detect the rapamycin-mediated interaction between FRAP and FKBP12 asdescribed above. Such experiments would show whether the rate ofdimerization was due to the kinetics of interaction of thenon-β-lactamase portions of the chimeric proteins. Such experimentswould also show that β-lactamase complementation can be used to monitorthe regulation of dimerization by other proteins.

[0152] Comparison to previous methods. Receptor dimerization hastypically been studied by in vitro methods such as chemicalcross-linking and immunopurification, followed by gel electrophoresis.Yarden et al. (1987) Biochemistry 26:1443-1451. Recently, EGF receptordimerization has also been analyzed by fluorescence resonance energytransfer (FRET). Gadella et al. (1995) supra. Fluorescein and rhodaminelabeled EGF was added to cells, and dimerization of the receptor wasmeasured microscopically. Low temperature incubations and fixation ofthe cells was required to prevent internalization of the receptor beforeanalysis, a problem that can be avoided by using a non-internalizingmutant receptor. FRET can also be used to study interactions offluorescently-labeled molecules within the cell or cell membrane;however, labeling and introduction of these molecules at sufficientlyhigh concentration can be cumbersome. It has recently been shown thatgreen fluorescent protein can be modified and used for FRET analysis ongenetically expressed proteins. Miyawaki et al. (1997) Nature388:882-887. However, the GFP signal, however, cannot be enzymaticallyamplified as is the case with β-galactosidase (Blakely et al., Nat.Biotechnol. 18:218-222 (2000)) or β-lactamase.

[0153] Thus, β-lactamase complementation provides a rapid method formonitoring receptor dimerization in live cells. This method can be usedfor high throughput screening for pharmacological agents that can bindto a number of receptors and act as either agonists or antagonists.Binding data alone cannot indicate whether or not an agent can elicit aresponse; identifying a response, by analysis of downstream effects suchas phosphorylation, involves destruction of the cells followed by invitro analysis. β-lactamase complementation will also enable a screenfor novel dimerization partners in a mammalian “two-hybrid” assay that,in the case of membrane receptors, can offer new insight into theregulation of signal transduction pathways.

[0154] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore theforegoing descriptions and examples should not be construed as limitingthe scope of the invention.

[0155] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A reporter system for detecting molecularinteractions in a eukaryotic cell or lysate thereof, the systemcomprising: a first low-affinity beta-lactamase fragment coupled to afirst putative binding moiety, wherein the first low-affinitybeta-lactamase fragment associates with at least a second low-affinitybeta-lactamase fragment to generate an enzymatically active complex,said association being mediated by the first putative binding moiety;and at least the second low-affinity beta-lactamase fragment coupled toa second putative binding moiety capable of binding to the firstputative binding moiety, wherein such binding generates theenzymatically active complex.
 2. The reporter system of claim 1, whereinthe binding affinity of the first and second putative binding moietiesfor each other is greater than the binding affinity of the first andsecond beta-lactamase fragments for each other.
 3. The reporter systemof claim 1, wherein the first and second putative binding moieties areproteins.
 4. The reporter system of claim 3, wherein the protein isselected from the group consisting of members of a signal transductioncascade, cell surface receptors, proteins regulating apoptosis, proteinsthat regulate progression of the cell-cycle, proteins involved in thedevelopment of tumors, transcriptional-regulatory proteins,translational regulatory proteins, proteins that affect cellinteractions, cell adhesion molecules, proteins which are members ofligand-receptor pairs, proteins that participate in the folding of otherproteins, and proteins involved in targeting to intracellularcompartments.
 5. The reporter system of claim 3, wherein the first andsecond beta-lactamase fragments are capable of associating to catalyze areaction to produce a detectable signal.
 6. The reporter system of claim5 wherein the associated beta-lactamase fragments catalyze a reaction toproduce a product which is directly detectable as the detectable signal.7. The reporter system of claim 5 wherein the detectable signal isamplifiable.
 8. The reporter system of claim 5 wherein the detectablesignal is generated in situ in the cell.
 9. The reporter system of claim1, wherein the first low-affinity beta-lactamase fragment and the firstputative binding moiety comprises a fusion protein.
 10. The reportersystem of claim 1, wherein the first low-affinity beta-lactamasefragment and the first putative binding moiety and the secondlow-affinity beta-lactamase fragment and the second putative bindingmoiety each comprise a fusion protein.
 11. The reporter system of claim1, wherein the eukaryotic cell is a mammalian cell.
 12. The reportersystem of claim 1, wherein the first and second low-affinitybeta-lactamase fragments comprise α197 and ω198 fragments ofbeta-lactamase.
 13. The reporter system of claim 12, wherein the firstand second putative binding moieties form a leucine zipper.
 14. Anucleic acid encoding the fusion protein of claim
 9. 15. The nucleicacid of claim 14 further comprising regulatory sequences effectingexpression of the putative binding protein.
 16. A viral vectorcomprising the nucleic acid of claim
 14. 17. A eukaryotic celltransformed with the nucleic acid of claim
 14. 18. An eukaryotic celltransformed with a first nucleic acid encoding the fusion protein ofclaim 9 and a second nucleic acid encoding a fusion protein comprisingthe second low-affinity beta-lactamase fragment coupled to a secondputative binding moiety.
 19. The eukaryotic cell of claim 18, whereinthe eukaryotic cell is a mammalian cell.
 20. The fusion protein of claim9, wherein the fusion protein further comprises an additional proteinsequence between the beta-lactamase fragment and the putative bindingmoiety.
 21. A method for determining the occurrence of binding between afirst and a second putative binding moieties, the method comprising: a)providing a reporter system comprising: a first component comprising afirst low-affinity beta-lactamase fragment coupled to a first putativebinding moiety, wherein the first low-affinity beta-lactamase fragmentassociates with at least a second low-affinity beta-lactamase fragmentto generate an enzymatically active complex, said association beingmediated by the first putative binding moiety; and a second componentcomprising at least the second low-affinity beta-lactamase fragmentcoupled to a second putative binding moiety capable of binding to thefirst putative binding moiety, wherein such binding generates theenzymatically active complex; b) combining the first component and thesecond component; and c) detecting the presence or absence of enzymeactivity.
 22. The method of claim 21 wherein the binding affinity of thefirst and second putative binding moieties for each other is greaterthan the binding affinity of the first and second beta-lactamasefragments for each other.
 23. The method of claim 22 wherein the firstand second putative binding moieties are proteins.
 24. The method ofclaim 23 wherein the protein is selected from the group consisting ofmembers of a signal transduction cascade, cell surface receptors,proteins regulating apoptosis, proteins that regulate progression of thecell-cycle, proteins involved in the development of tumors,transcriptional-regulatory proteins, translation regulatory proteins,proteins that affect cell interactions, cell adhesion molecules,proteins which are members of ligand-receptor pairs, proteins thatparticipate in the folding of other proteins, and proteins involved intargeting to intracellular compartments.
 25. The method of claim 21,wherein the first and second beta-lactamase fragment are capable ofassociating to catalyze a reaction to produce a detectable signal. 26.The method of claim 25, wherein the associated beta-lactamase fragmentcatalyze a reaction to produce a product which is directly detectable asthe detectable signal.
 27. The method of claim 25, wherein thedetectable signal is amplifiable.
 28. The method of claim 25, whereinthe detectable signal is generated in situ in the cell.
 29. The methodof claim 21, wherein each of said first and second components comprisesa fusion protein.
 30. The method of claim 29 wherein step (a) comprisestransforming a cell with one or more nucleic acids encoding the fusionproteins.
 31. The method of claim 30 wherein step (c) comprisesdetecting the presence or absence of enzymatic activity within the cell.32. The method of claim 30 wherein the one or more nucleic acidsencoding the fusion proteins further comprise sequences regulatingexpression of the putative binding protein.
 33. The method of claim 30wherein the fusion proteins are encoded by a viral vector.
 34. Themethod of claim 29 wherein the fusion protein further comprises aprotein sequence between said beta-lactamase fragment and said putativebinding moiety.
 35. The method of claim 21, wherein the method furthercomprises: (d) detecting the effect of a third moiety on the binding ofthe first and second binding moieties by combining said reporter systemwith said third moiety following step (a) and prior to step (b).
 36. Themethod of claim 31 wherein the intracellular localization of theenzymatic activity is determined.
 37. The method of claim 21, whereinstep (b) comprises combining the first and second components in thepresence of a substance to determine the effect of the substance onbinding of the first and second binding moieties.
 38. The method ofclaim 37 wherein the substance is a peptide, drug or synthetic analog.39. The method of claim 37 wherein the substance is a putative inhibitorof binding between binding moieties having a predetermined bindingaffinity, and wherein the absence of enzymatic activity in step (c)indicates that the substance is a binding inhibitor.
 40. The method ofclaim 37 wherein the substance is a putative promoter of binding betweenbinding moieties having low or substantially no binding affinity foreach other, and wherein the presence of enzymatic activity in step (c)provides an indicator that the substance is a promoter of binding of thebinding moieties.
 41. The method of claim 37, wherein the substancedirectly or indirectly affects an upstream event which results in aneffect on binding of the first and second binding moieties.
 42. Themethod of claim 37 wherein the first and second binding moieties areproteins; the first and second components of step (a) each comprise afusion protein; step (b) comprises expressing nucleic acid sequencesencoding the first and second components within a cell suspected tocontain the substance which inhibits or promotes binding of the bindingmoieties; and step (c) comprises detecting the presence or absence ofenzymatic activity in the cell or a lysate thereof which correlates witha presence or absence in the cell of the substance which is a promoteror an inhibitor of binding between the binding moieties.
 43. The methodof claim 37 wherein the substance is selected from the group consistingof a protein, lipid, carbohydrate, nucleic acid, and a small moleculepharmaceutical.
 44. A method of screening for binding of a first bindingmoiety with members of a plurality of different second putative bindingmoieties, the method comprising: a) providing a plurality of reportersystems each comprising: a first component comprising a first lowaffinity beta-lactamase fragment coupled to a first binding moiety, andone of a plurality of second components each comprising a second lowaffinity beta-lactamase fragment coupled to one of said plurality ofsecond putative binding moieties, wherein in each of said secondcomponents, said second putative binding moiety is different, andfurther wherein the first low affinity beta-lactamase fragmentassociates with the second low affinity beta-lactamase fragment togenerate an enzymatically active complex upon the binding of the firstbinding moiety with one of said different second putative bindingmoieties; b) individually combining the first component with each of theplurality of second components to produce a plurality of binding assaysamples, each of which includes the first component and a different oneof the second components; and c) detecting the presence or absence ofenzymatic activity in each of the binding assay samples.
 45. The methodof claim 44, wherein the first and second components each comprise afusion protein including the binding moiety and the beta-lactamasefragment.
 46. The method of claim 45, wherein, in step (b), thecomponents are expressed from a nucleic acid sequence introduced into acell.
 47. The method of claim 46, wherein the plurality of secondputative binding moieties are encoded by members of a cDNA library. 48.The method of claim 46, wherein the cell is a mammalian cell.
 49. Themethod of claim 48, wherein the cell is a human cell.
 50. The method ofclaim 44, wherein, in step (c), enzymatic activity is quantitated. 51.The method of claim 44, wherein the first and second beta-lactamasefragment are capable of associating to catalyze a reaction to produce adetectable signal.
 52. The method of claim 51, wherein the associatedbeta-lactamase fragment catalyze a reaction to produce a product whichis directly detectable as the detectable signal.
 53. The method of claim51, wherein the detectable signal is amplifiable.
 54. The method ofclaim 51, wherein the detectable signal is generated in situ in thecell.
 55. The method of claim 44, wherein cells in which binding betweenthe first binding moiety and one of the plurality of putative secondbinding moieties has occurred are separated from cells in which saidbinding has not occurred.
 56. The method of claim 55, wherein separationis by fluorescence-activated cell sorting.
 57. The method of claim 44,wherein the first binding moiety is selected from the group consistingof cell surface receptors, transcriptional regulatory proteins,translation regulatory proteins, replication proteins, splicingproteins, signal transduction proteins, cell-cell adhesion molecules,cell-substrate adhesion molecules, cell-cycle proteins, oncogeneproducts, tumor suppressor proteins, membrane receptors, proteinsregulating apoptosis, developmental regulatory proteins, proteins thataffect cell interactions, proteins that participate in the folding ofother proteins, proteins involved in targeting to intracellularcompartments, viral proteins, and cytoskeletal proteins.